X-linked genetic associations in sporadic thoracic aortic dissection.

The male predominance in sporadic thoracic aortic aneurysm and dissection (TAD) suggests that the X chromosome contributes to TAD, but this has not been tested. We investigated whether X-linked variation-common (minor allele frequency [MAF] ≥0.01) and rare (MAF <0.01)-was associated with sporadic TAD in three cohorts of European descent (Discovery: 364 cases, 874 controls; Replication: 516 cases, 440,131 controls, and ARIC [Atherosclerosis Risk in Communities study]: 753 cases, 2247 controls). For analysis of common variants, we applied a sex-stratified logistic regression model followed by a meta-analysis of sex-specific odds ratios. Furthermore, we conducted a meta-analysis of overlapping common variants between the Discovery and Replication cohorts. For analysis of rare variants, we used a sex-stratified optimized sequence kernel association test model. Common variants results showed no statistically significant findings in the Discovery cohort. An intergenic common variant near SPANXN1 was statistically significant in the Replication cohort (p = 1.81 × 10-8). The highest signal from the meta-analysis of the Discovery and Replication cohorts was a ZNF182 intronic common variant (p = 3.5 × 10-6). In rare variants results, RTL9 reached statistical significance (p = 5.15 × 10-5). Although most of our results were statistically insignificant, our analysis is the most comprehensive X-chromosome association analysis of sporadic TAD to date.

A Compound that Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer.

Metastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Non-specific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. We tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell cycle, metabolic and enzymatic assays were used to demonstrate their mechanism of action. A human PDX model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. We demonstrate a new class of small molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. This work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.

Dynamic Prediction of Advanced Colorectal Neoplasia in Inflammatory Bowel Disease.

BACKGROUND & AIMS: Colonoscopic surveillance is recommended in patients with colonic inflammatory bowel disease (IBD) given their increased risk of colorectal cancer (CRC). We aimed to develop and validate a dynamic prediction model for the occurrence of advanced colorectal neoplasia (aCRN, including high-grade dysplasia and CRC) in IBD. METHODS: We pooled data from 6 existing cohort studies from Canada, The Netherlands, the United Kingdom, and the United States. Patients with IBD and an indication for CRC surveillance were included if they underwent at least 1 follow-up procedure. Exclusion criteria included prior aCRN, prior colectomy, or an unclear indication for surveillance. Predictor variables were selected based on the literature. A dynamic prediction model was developed using a landmarking approach based on Cox proportional hazard modeling. Model performance was assessed with Harrell's concordance-statistic (discrimination) and by calibration curves. Generalizability across surveillance cohorts was evaluated by internal-external cross-validation. RESULTS: The surveillance cohorts comprised 3731 patients, enrolled and followed-up in the time period from 1973 to 2021, with a median follow-up period of 5.7 years (26,336 patient-years of follow-up evaluation); 146 individuals were diagnosed with aCRN. The model contained 8 predictors, with a cross-validation median concordance statistic of 0.74 and 0.75 for a 5- and 10-year prediction window, respectively. Calibration plots showed good calibration. Internal-external cross-validation results showed medium discrimination and reasonable to good calibration. CONCLUSIONS: The new prediction model showed good discrimination and calibration, however, generalizability results varied. Future research should focus on formal external validation and relate predicted aCRN risks to surveillance intervals before clinical application.

Characterization of an Acinetobacter baumannii Monofunctional Phosphomethylpyrimidine Kinase That Is Inhibited by Pyridoxal Phosphate.

Thiamin and its phosphate derivatives are ubiquitous molecules involved as essential cofactors in many cellular processes. The de novo biosynthesis of thiamin employs the parallel synthesis of 4-methyl-5-(2-hydroxyethyl)thiazole (THZ-P) and 4-amino-2-methyl-5(diphosphooxymethyl) pyrimidine (HMP) pyrophosphate (HMP-PP), which are coupled to generate thiamin phosphate. Most organisms that can biosynthesize thiamin employ a kinase (HMPK or ThiD) to generate HMP-PP. In nearly all cases, this enzyme is bifunctional and can also salvage free HMP, producing HMP-P, the monophosphate precursor of HMP-PP. Here we present high-resolution crystal structures of an HMPK from Acinetobacter baumannii (AbHMPK), both unliganded and with pyridoxal 5-phosphate (PLP) noncovalently bound. Despite the similarity between HMPK and pyridoxal kinase enzymes, our kinetics analysis indicates that AbHMPK accepts HMP exclusively as a substrate and cannot turn over pyridoxal, pyridoxamine, or pyridoxine nor does it display phosphatase activity. PLP does, however, act as a weak inhibitor of AbHMPK with an IC50 of 768 µM. Surprisingly, unlike other HMPKs, AbHMPK catalyzes only the phosphorylation of HMP and does not generate the diphosphate HMP-PP. This suggests that an additional kinase is present in A. baumannii, or an alternative mechanism is in operation to complete the biosynthesis of thiamin.

A Case of Whipple's Disease With Concomitant Esophageal Candidiasis.

Whipple's Disease (WD) is a rare disease caused by the infection of Tropheryma whipplei. It can lead to immunosuppression and a multitude of effects on different organ systems, resulting in a constellation of seemingly unrelated findings. Although treatment may appear straightforward, T. whipplei can be difficult to eradicate. We present the case of a 36-year-old male with months of progressively worsening watery diarrhea, migratory arthralgias, and weight loss. He had undergone an extensive evaluation for rheumatologic, oncologic, and infectious disorders without positive findings. Esophagogastroduodenoscopy and colonoscopy revealed esophageal candidiasis, Helicobacter pylori infection, and foamy macrophages in the lamina propria of the duodenum and ileum with positive polymerase chain reaction for T. whipplei. There were no other risk factors for esophageal candidiasis. He received treatment for his esophageal candidiasis and H. pylori infection and was treated for WD with ceftriaxone for 2 weeks, followed by hydroxychloroquine and doxycycline for 1 year. Symptoms resolved after 3 months of therapy. One year later, repeat bidirectional endoscopy was performed. Biopsies were negative for T. whipplei, although there were persistent foamy macrophages. There have been previously reported cases of patients with WD with concomitant esophageal candidiasis, and this association implies a likely state of relative immunosuppression associated with WD, which is thought to be the result of impaired T helper cell 1 activity. This impairment likely contributes to the high rate of relapse. Having a low threshold for repeat evaluation is advisable for recurrent symptoms, but long-term surveillance strategies are not clearly defined.

A Compound that Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer.

Purpose: Metastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Non-specific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. Experimental design: We tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell cycle, metabolic and enzymatic assays were used to demonstrate their mechanism of action. A human PDX model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. Results: We demonstrate a new class of small molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. Conclusion: This work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.

An Early Treatment With BKI-1748 Exhibits Full Protection Against Abortion and Congenital Infection in Sheep Experimentally Infected With Toxoplasma gondii.

Congenital toxoplasmosis in humans and in other mammalian species, such as small ruminants, is a well-known cause of abortion and fetal malformations. The calcium-dependent protein kinase 1 (CDPK1) inhibitor BKI-1748 has shown a promising safety profile for its use in humans and a good efficacy against Toxoplasma gondii infection in vitro and in mouse models. Ten doses of BKI-1748 given every other day orally in sheep at 15 mg/kg did not show systemic or pregnancy-related toxicity. In sheep experimentally infected at 90 days of pregnancy with 1000 TgShSp1 oocysts, the BKI-1748 treatment administered from 48 hours after infection led to complete protection against abortion and congenital infection. In addition, compared to infected/untreated sheep, treated sheep showed a drastically lower rectal temperature increase and none showed IgG seroconversion throughout the study. In conclusion, BKI-1748 treatment in pregnant sheep starting at 48 hours after infection was fully effective against congenital toxoplasmosis.

Regulation of coronavirus nsp15 cleavage specificity by RNA structure.

SARS-CoV-2, the etiologic agent of the COVID-19 pandemic, has had an enduring impact on global public health. However, SARS-CoV-2 is only one of multiple pathogenic human coronaviruses (CoVs) to have emerged since the turn of the century. CoVs encode for several nonstructural proteins (nsps) that are essential for viral replication and pathogenesis. Among them is nsp15, a uridine-specific viral endonuclease that is important in evading the host immune response and promoting viral replication. Despite the established endonuclease function of nsp15, little is known about other determinants of its cleavage specificity. In this study we investigate the role of RNA secondary structure in SARS-CoV-2 nsp15 endonuclease activity. Using a series of in vitro endonuclease assays, we observed that thermodynamically stable RNA structures were protected from nsp15 cleavage relative to RNAs lacking stable structure. We leveraged the s2m RNA from the SARS-CoV-1 3'UTR as a model for our structural studies as it adopts a well-defined structure with several uridines, two of which are unpaired and thus highly probable targets for nsp15 cleavage. We found that SARS-CoV-2 nsp15 specifically cleaves s2m at the unpaired uridine within the GNRNA pentaloop of the RNA. Further investigation revealed that the position of uridine within the pentaloop also impacted nsp15 cleavage efficiency suggesting that positioning within the pentaloop is necessary for optimal presentation of the scissile uridine and alignment within the nsp15 catalytic pocket. Our findings indicate that RNA secondary structure is an important determinant of nsp15 cleavage and provides insight into the molecular mechanisms of RNA recognition by nsp15.

Identification of Fungus-Derived Natural Products as New Antigiardial Scaffolds.

There is an unmet need for effective therapies for treating diseases associated with the intestinal parasite Giardia lamblia. In this study, a library of chemically validated purified natural products and fungal extracts was screened for chemical scaffolds that can inhibit the growth of G. lamblia. The phenotypic screen led to the identification of several previously unreported classes of natural product inhibitors that block the growth of G. lamblia. Hits from phenotypic screens of these naturally derived compounds are likely to possess a variety of mechanisms of action not associated with clinically used nitroimidazole and thiazolide compounds. They may therefore be effective against current drug-resistant parasite strains. IMPORTANCE There is a direct link between widespread prevalence of clinical giardiasis and poverty. This may be one of the reasons why giardiasis is a significant contributor to diarrheal morbidity, stunting, and death of children in resource-limited communities around the world. FDA-approved treatments for giardiasis include metronidazole, related nitroimidazole drugs, and albendazole. However, a substantial number of clinical infections are resistant to these treatments. The depth of the challenge is partly exacerbated by a lack of investment in the discovery and development of novel agents for treatment of giardiasis. Applicable interventions must include new drug development strategies that will result in the identification of effective therapeutics, particularly those that are inexpensive and can be quickly advanced to clinical uses, such as products from nature. This study identified novel chemical scaffolds from fungi that can form the basis of future medicinal chemistry optimization of novel antigiardial agents.

Comparison of Toxicities among Different Bumped Kinase Inhibitor Analogs for Treatment of Cryptosporidiosis.

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. Both BKI-1770 and BKI-1841 had efficacy in the C. parvum newborn calf model, reducing diarrhea and oocyst excretion. However, both compounds caused hyperflexion of the limbs seen as dropped pasterns. Toxicity experiments in rats and calves dosed with BKI-1770 showed enlargement of the epiphyseal growth plate at doses only slightly higher than the efficacious dose. Mice were used as a screen to check for bone toxicity, by changes to the tibia epiphyseal growth plate, or neurological causes, by use of a locomotor activity box. These results showed neurological effects from both BKI-1770 and BKI-1841 and bone toxicity in mice from BKI-1770, indicating one or both effects may be contributing to toxicity. However, BKI-1708 remains a viable treatment candidate for further evaluation as it showed no signs of bone toxicity or neurological effects in mice.

Accurate de novo design of membrane-traversing macrocycles.

We use computational design coupled with experimental characterization to systematically investigate the design principles for macrocycle membrane permeability and oral bioavailability. We designed 184 6-12 residue macrocycles with a wide range of predicted structures containing noncanonical backbone modifications and experimentally determined structures of 35; 29 are very close to the computational models. With such control, we show that membrane permeability can be systematically achieved by ensuring all amide (NH) groups are engaged in internal hydrogen bonding interactions. 84 designs over the 6-12 residue size range cross membranes with an apparent permeability greater than 1 × 10-6 cm/s. Designs with exposed NH groups can be made membrane permeable through the design of an alternative isoenergetic fully hydrogen-bonded state favored in the lipid membrane. The ability to robustly design membrane-permeable and orally bioavailable peptides with high structural accuracy should contribute to the next generation of designed macrocycle therapeutics.

Repurposing the Kinase Inhibitor Mavelertinib for Giardiasis Therapy.

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 in vitro efficacy against metronidazole-resistant 713-M3 strains. Other EGFR-TKIs screened in follow-up assays exhibited insignificant inhibition of G. lamblia at 5 µM, suggesting that the primary molecular target of mavelertinib may have a different mechanistic binding mode from human EGFR-tyrosine kinase. Mavelertinib, dosed as low as 5 mg/kg of body weight or as high as 50 mg/kg, was efficacious in the acute murine Giardia infection model. These results suggest that mavelertinib merits consideration for repurposing and advancement to giardiasis clinical trials while its analogues are further developed.

Multiple resource limitation of dryland soil microbial carbon cycling on the Colorado Plateau.

Understanding interactions among biogeochemical cycles is increasingly important as anthropogenic alterations of global climate and of carbon (C), nitrogen (N), and phosphorus (P) cycles interactively affect the Earth system. Ecosystem processes in the dryland biome, which makes up over 40% of Earth's terrestrial surface, are often distinctively sensitive to small changes in resource availability, likely because levels of many resources are low. However, data also suggest that simultaneous changes in the availability of multiple resources may be necessary to affect a response in these low-resource systems, offering an opportunity to test patterns and controls of co-limitation, serial limitation, and individual limitation in soil environments. While drylands may play a governing role in key aspects of Earth's C cycle, and while an improved understanding of resource limitation could substantially improve our forecasts of dryland responses to change, our understanding of interacting controls on soil C cycle processes remains notably poor in these dry systems. Here, we address multiple fundamental hypotheses of resource controls over ecosystem function to test how water, C, N, and P regulate soil C cycling individually and interactively in a dryland ecosystem on the Colorado Plateau. Using a series of laboratory incubations, we found that, while water, C, and N limited C cycling through serial limitation, water alone resulted in an extremely small respiratory response from target organisms, whereas water + C resulted in a dramatic increase in soil C cycling, suggesting a degree of functional co-limitation. Nitrogen additions alone resulted in no changes to soil C cycling, but when N was added in concert with water and C, N greatly increased soil C cycling rates relative to additions of water and C without N. Phosphorus additions had no effect on the C cycle either alone or synergistically. These patterns were consistent with the stoichiometry of the system and interactions among resources were surprising in ways that inform our understanding of critical theories in ecology, such as the Transient Maxima Hypothesis, supporting the suggestion that multiple resource limitation explains pulse-dynamic C cycling in drylands better than water limitation alone.

A short-term treatment with BKI-1294 does not protect foetuses from sheep experimentally infected with Neospora caninum tachyzoites during pregnancy.

The Neospora caninum Calcium-dependent protein kinase 1 (NcCDPK1) inhibitor BKI-1294 had demonstrated excellent efficacy in a pregnant mouse model of neosporosis, and was also highly efficacious in a pregnant sheep model of toxoplasmosis. In this work, we present the efficacy of BKI-1294 treatment (dosed 5 times orally every 48 h) starting 48 h after intravenous infection of sheep with 105 Nc-Spain7 tachyzoites at mid-pregnancy. In the dams, BKI-1294 plasma concentrations were above the IC50 for N. caninum for 12-15 days. In treated sheep, when they were compared to untreated ones, we observed a minor increase in rectal temperature, higher IFNγ levels after blood stimulation in vitro, and a minor increase of IgG levels against N. caninum soluble antigens through day 28 post-infection. Additionally, the anti-NcSAG1 and anti-NcSAG4 IgGs were lower in treated dams on days 21 and 42 post-infection. However, BKI-1294 did not protect against abortion (87% foetal mortality in both infected groups, treated and untreated) and did not reduce transplacental transmission, parasite load or lesions in placentomes and foetal brain. The lack of foetal protection was likely caused by short systemic exposure in the dams and suboptimal foetal exposure to this parasitostatic drug, which was unable to reduce replication of the likely established N. caninum tachyzoites in the foetus at the moment of treatment. New BKIs with a very low plasma clearance and good ability to cross the blood-brain and placental barriers need to be developed.

Endochin-like quinolones (ELQs) and bumped kinase inhibitors (BKIs): Synergistic and additive effects of combined treatments against Neospora caninum infection in vitro and in vivo.

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.

In vitro activity, safety and in vivo efficacy of the novel bumped kinase inhibitor BKI-1748 in non-pregnant and pregnant mice experimentally infected with Neospora caninum tachyzoites and Toxoplasma gondii oocysts.

Bumped kinase inhibitors (BKIs) target the apicomplexan calcium-dependent protein kinase 1 (CDPK1). BKI-1748, a 5-aminopyrazole-4-carboxamide compound when added to fibroblast cells concomitantly to the time of infection, inhibited proliferation of apicomplexan parasites at EC50s of 165 nM (Neospora caninum) and 43 nM (Toxoplasma gondii). Immunofluorescence and electron microscopy showed that addition of 2.5 µM BKI-1748 to infected HFF monolayers transformed parasites into multinucleated schizont-like complexes (MNCs) containing newly formed zoites, which were unable to separate and form infective tachyzoites or undergo egress. In zebrafish (Danio rerio) embryo development assays, no embryonic impairment was detected within 96 h at BKI-1748 concentrations up to 10 µM. In pregnant mice, BKI-1748 applied at days 9-13 of pregnancy at a dose of 20 mg/kg/day was safe and no pregnancy interference was observed. The efficacy of BKI-1748 was assessed in standardized pregnant mouse models infected with N. caninum (NcSpain-7) tachyzoites or T. gondii (TgShSp1) oocysts. In both models, treatments resulted in increased pup survival and profound inhibition of vertical transmission. However, in dams and non-pregnant mice, BKI-1748 treatments resulted in significantly decreased cerebral parasite loads only in T. gondii infected mice. In the T. gondii-model, ocular infection was detected in 10 out of 12 adult mice of the control group, but only in 3 out of 12 mice in the BKI-1748-treated group. Thus, TgShSp1 oocyst infection is a suitable model to study both cerebral and ocular infection by T. gondii. BKI-1748 represents an interesting candidate for follow-up studies on neosporosis and toxoplasmosis in larger animal models.

High-throughput screening of the ReFRAME, Pandemic Box, and COVID Box drug repurposing libraries against SARS-CoV-2 nsp15 endoribonuclease to identify small-molecule inhibitors of viral activity.

SARS-CoV-2 has caused a global pandemic, and has taken over 1.7 million lives as of mid-December, 2020. Although great progress has been made in the development of effective countermeasures, with several pharmaceutical companies approved or poised to deliver vaccines to market, there is still an unmet need of essential antiviral drugs with therapeutic impact for the treatment of moderate-to-severe COVID-19. Towards this goal, a high-throughput assay was used to screen SARS-CoV-2 nsp15 uracil-dependent endonuclease (endoU) function against 13 thousand compounds from drug and lead repurposing compound libraries. While over 80% of initial hit compounds were pan-assay inhibitory compounds, three hits were confirmed as nsp15 endoU inhibitors in the 1-20 µM range in vitro. Furthermore, Exebryl-1, a ß-amyloid anti-aggregation molecule for Alzheimer's therapy, was shown to have antiviral activity between 10 to 66 µM, in Vero 76, Caco-2, and Calu-3 cells. Although the inhibitory concentrations determined for Exebryl-1 exceed those recommended for therapeutic intervention, our findings show great promise for further optimization of Exebryl-1 as an nsp15 endoU inhibitor and as a SARS-CoV-2 antiviral.

Repurposing Infectious Disease Hits as Anti-Cryptosporidium Leads.

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.

Pyrrolopyrimidine Bumped Kinase Inhibitors for the Treatment of Cryptosporidiosis.

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.

One health therapeutics: Target-Based drug development for cryptosporidiosis and other apicomplexa diseases.

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.