Publications by authors named "Wesley C Van Voorhis"

207 Publications

Special Issue on Gut Pathogens.

ACS Infect Dis 2021 May 3;7(5):938. Epub 2021 May 3.

Global Health Pharma Unit, GlaxoSmithKline, Tres Cantos 28760, Spain.

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http://dx.doi.org/10.1021/acsinfecdis.1c00214DOI Listing
May 2021

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.

PLoS One 2021 22;16(4):e0250019. Epub 2021 Apr 22.

Division of Allergy and Infectious Diseases, Department of Medicine, Center for Emerging and Reemerging Infectious Diseases (CERID), University of Washington School of Medicine, Seattle, WA, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0250019PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062000PMC
May 2021

SARS-CoV-2 immune evasion by variant B.1.427/B.1.429.

bioRxiv 2021 Apr 1. Epub 2021 Apr 1.

SARS-CoV-2 entry is mediated by the spike (S) glycoprotein which contains the receptor-binding domain (RBD) and the N-terminal domain (NTD) as the two main targets of neutralizing antibodies (Abs). A novel variant of concern (VOC) named CAL.20C (B.1.427/B.1.429) was originally detected in California and is currently spreading throughout the US and 29 additional countries. It is unclear whether antibody responses to SARS-CoV-2 infection or to the prototypic Wuhan-1 isolate-based vaccines will be impacted by the three B.1.427/B.1.429 S mutations: S13I, W152C and L452R. Here, we assessed neutralizing Ab responses following natural infection or mRNA vaccination using pseudoviruses expressing the wildtype or the B.1.427/B.1.429 S protein. Plasma from vaccinated or convalescent individuals exhibited neutralizing titers, which were reduced 3-6 fold against the B.1.427/B.1.429 variant relative to wildtype pseudoviruses. The RBD L452R mutation reduced or abolished neutralizing activity of 14 out of 35 RBD-specific monoclonal antibodies (mAbs), including three clinical-stage mAbs. Furthermore, we observed a complete loss of B.1.427/B.1.429 neutralization for a panel of mAbs targeting the N-terminal domain due to a large structural rearrangement of the NTD antigenic supersite involving an S13I-mediated shift of the signal peptide cleavage site. These data warrant closer monitoring of signal peptide variants and their involvement in immune evasion and show that Abs directed to the NTD impose a selection pressure driving SARS-CoV-2 viral evolution through conventional and unconventional escape mechanisms.
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http://dx.doi.org/10.1101/2021.03.31.437925DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8020983PMC
April 2021

Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba.

PLoS One 2021 24;16(3):e0241738. Epub 2021 Mar 24.

Department of Biology, Washington University, St. Louis, Missouri, United States of America.

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain's frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen's high mortality is the lack of sensitivity of N. fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N. fowleri. The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N. fowleri research. In 2017, 178 N. fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0241738PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7990177PMC
March 2021

Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines.

bioRxiv 2021 Mar 16. Epub 2021 Mar 16.

Understanding the ability of SARS-CoV-2 vaccine-elicited antibodies to neutralize and protect against emerging variants of concern and other sarbecoviruses is key for guiding vaccine development decisions and public health policies. We show that a clinical stage multivalent SARS-CoV-2 receptor-binding domain nanoparticle vaccine (SARS-CoV-2 RBD-NP) protects mice from SARS-CoV-2-induced disease after a single shot, indicating that the vaccine could allow dose-sparing. SARS-CoV-2 RBD-NP elicits high antibody titers in two non-human primate (NHP) models against multiple distinct RBD antigenic sites known to be recognized by neutralizing antibodies. We benchmarked NHP serum neutralizing activity elicited by RBD-NP against a lead prefusion-stabilized SARS-CoV-2 spike immunogen using a panel of single-residue spike mutants detected in clinical isolates as well as the B.1.1.7 and B.1.351 variants of concern. Polyclonal antibodies elicited by both vaccines are resilient to most RBD mutations tested, but the E484K substitution has similar negative consequences for neutralization, and exhibit modest but comparable neutralization breadth against distantly related sarbecoviruses. We demonstrate that mosaic and cocktail sarbecovirus RBD-NPs elicit broad sarbecovirus neutralizing activity, including against the SARS-CoV-2 B.1.351 variant, and protect mice against severe SARS-CoV challenge even in the absence of the SARS-CoV RBD in the vaccine. This study provides proof of principle that sarbecovirus RBD-NPs induce heterotypic protection and enables advancement of broadly protective sarbecovirus vaccines to the clinic.
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http://dx.doi.org/10.1101/2021.03.15.435528DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986998PMC
March 2021

Repurposing Infectious Disease Hits as Anti- Leads.

ACS Infect Dis 2021 May 19;7(5):1275-1282. Epub 2021 Mar 19.

Department of Medicine Division of Allergy Infectious Disease Center for Emerging Reemerging Infectious Diseases, University of Washington, Seattle, Washington 98109, United States.

New drugs are critically needed to treat 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 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 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 was not definitively established, this imidazole-pyrimidine compound inhibits the known 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 therapeutics.
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http://dx.doi.org/10.1021/acsinfecdis.1c00076DOI Listing
May 2021

In silico detection of SARS-CoV-2 specific B-cell epitopes and validation in ELISA for serological diagnosis of COVID-19.

Sci Rep 2021 02 22;11(1):4290. Epub 2021 Feb 22.

Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, WA, USA.

Rapid generation of diagnostics is paramount to understand epidemiology and to control the spread of emerging infectious diseases such as COVID-19. Computational methods to predict serodiagnostic epitopes that are specific for the pathogen could help accelerate the development of new diagnostics. A systematic survey of 27 SARS-CoV-2 proteins was conducted to assess whether existing B-cell epitope prediction methods, combined with comprehensive mining of sequence databases and structural data, could predict whether a particular protein would be suitable for serodiagnosis. Nine of the predictions were validated with recombinant SARS-CoV-2 proteins in the ELISA format using plasma and sera from patients with SARS-CoV-2 infection, and a further 11 predictions were compared to the recent literature. Results appeared to be in agreement with 12 of the predictions, in disagreement with 3, while a further 5 were deemed inconclusive. We showed that two of our top five candidates, the N-terminal fragment of the nucleoprotein and the receptor-binding domain of the spike protein, have the highest sensitivity and specificity and signal-to-noise ratio for detecting COVID-19 sera/plasma by ELISA. Mixing the two antigens together for coating ELISA plates led to a sensitivity of 94% (N = 80 samples from persons with RT-PCR confirmed SARS-CoV-2 infection), and a specificity of 97.2% (N = 106 control samples).
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http://dx.doi.org/10.1038/s41598-021-83730-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7900118PMC
February 2021

Pyrrolopyrimidine Bumped Kinase Inhibitors for the Treatment of Cryptosporidiosis.

ACS Infect Dis 2021 May 10;7(5):1200-1207. Epub 2021 Feb 10.

Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Re-emerging Infectious Disease (CERID), University of Washington, Seattle, Washington 98109, United States.

Bumped kinase inhibitors (BKIs) that target calcium-dependent protein kinase 1 have been well established as potential drug candidates against cryptosporidiosis. Recently, BKI-1649, with a 7-pyrrolo[2,3-]pyrimidin-4-amine, or "pyrrolopyrimidine", central scaffold, has shown improved efficacy in mouse models of 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.
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http://dx.doi.org/10.1021/acsinfecdis.0c00803DOI Listing
May 2021

Development of a target identification approach using native mass spectrometry.

Sci Rep 2021 Jan 27;11(1):2387. Epub 2021 Jan 27.

Griffith Institute for Drug Discovery, Griffith University, Brisbane, QLD, 4111, Australia.

A key step in the development of new pharmaceutical drugs is the identification of the molecular target and distinguishing this from all other gene products that respond indirectly to the drug. Target identification remains a crucial process and a current bottleneck for advancing hits through the discovery pipeline. Here we report a method, that takes advantage of the specific detection of protein-ligand complexes by native mass spectrometry (MS) to probe the protein partner of a ligand in an untargeted method. The key advantage is that it uses unmodified small molecules for binding and, thereby, it does not require labelled ligands and is not limited by the chemistry required to tag the molecule. We demonstrate the use of native MS to identify known ligand-protein interactions in a protein mixture under various experimental conditions. A protein-ligand complex was successfully detected between parthenolide and thioredoxin (PfTrx) in a five-protein mixture, as well as when parthenolide was mixed in a bacterial cell lysate spiked with PfTrx. We provide preliminary data that native MS could be used to identify binding targets for any small molecule.
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http://dx.doi.org/10.1038/s41598-021-81859-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7840913PMC
January 2021

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

Vet Parasitol 2021 Jan 15;289:109336. Epub 2020 Dec 15.

Center for Emerging and Re-emerging Infectious Diseases (CERID), Department of Medicine, Division of Allergy and Infectious Diseases, University of Washington, Seattle, WA, 98109, USA.

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.
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http://dx.doi.org/10.1016/j.vetpar.2020.109336DOI Listing
January 2021

Backbone chemical shift assignments for the SARS-CoV-2 non-structural protein Nsp9: intermediate (ms - μs) dynamics in the C-terminal helix at the dimer interface.

Biomol NMR Assign 2021 04 4;15(1):107-116. Epub 2021 Jan 4.

Seattle Structural Genomics Center for Infectious Disease, Seattle, USA.

The Betacoronavirus SARS-CoV-2 non-structural protein Nsp9 is a 113-residue protein that is essential for viral replication, and consequently, a potential target for the development of therapeutics against COVID19 infections. To capture insights into the dynamics of the protein's backbone in solution and accelerate the identification and mapping of ligand-binding surfaces through chemical shift perturbation studies, the backbone H, C, and N NMR chemical shifts for Nsp9 have been extensively assigned. These assignments were assisted by the preparation of an ~ 70% deuterated sample and residue-specific, N-labelled samples (V, L, M, F, and K). A major feature of the assignments was the "missing" amide resonances for N96-L106 in the H-N HSQC spectrum, a region that comprises almost the complete C-terminal α-helix that forms a major part of the homodimer interface in the crystal structure of SARS-CoV-2 Nsp9, suggesting this region either undergoes intermediate motion in the ms to μs timescale and/or is heterogenous. These "missing" amide resonances do not unambiguously appear in the H-N HSQC spectrum of SARS-CoV-2 Nsp9 collected at a concentration of 0.0007 mM. At this concentration, at the detection limit, native mass spectrometry indicates the protein is exclusively in the monomeric state, suggesting the intermediate motion in the C-terminal of Nsp9 may be due to intramolecular dynamics. Perhaps this intermediate ms to μs timescale dynamics is the physical basis for a previously suggested "fluidity" of the C-terminal helix that may be responsible for homophilic (Nsp9-Nsp9) and postulated heterophilic (Nsp9-Unknown) protein-protein interactions.
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http://dx.doi.org/10.1007/s12104-020-09992-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7779335PMC
April 2021

Prevalence and Correlates of Infections in Kenyan Children With Diarrhea and Their Primary Caregivers.

Open Forum Infect Dis 2020 Dec 31;7(12):ofaa533. Epub 2020 Oct 31.

University of Washington, Seattle, Washington, USA.

Background: is a leading cause of diarrhea in Sub-Saharan Africa and is associated with substantial morbidity and mortality in young children.

Methods: We analyzed data from children aged 6-71 months presenting to 2 public hospitals in Western Kenya with acute diarrhea and their primary caregivers, including detection of by quantitative polymerase chain reaction (PCR) and immunoassay analysis in stool samples from both children and their caregivers. Associations between potential transmission sources and child/caregiver infection were evaluated using prevalence ratios (PRs). Secondary analyses evaluated host and clinical risk factors of child/caregiver infection.

Results: Among 243 child-caregiver pairs enrolled, 77 children (32%) and 57 caregivers (23%) had identified by either immunoassay or PCR. Twenty-six of the 243 child-caregiver pairs (11%) had concordant detection of . infection in children was associated with detection of in caregivers (adjusted PR [aPR], 1.8; 95% CI, 1.2 to 2.6; .002) and unprotected water source (aPR, 2.0; 95% CI, 1.3 to 3.2; .003). Risk factors for detection in caregivers included child infection (aPR, 2.0; 95% CI, 1.3 to 3.0; .002) as well as cow (aPR, 3.1; 95% CI, 1.4 to 7.0; .02) and other livestock ownership (aPR, 2.6; 95% CI, 1.1 to 6.3; .03) vs no livestock ownership. Recent diarrhea in caregivers and children was independently associated with child and caregiver infections, respectively.

Conclusions: Our results are consistent with the hypothesis that transmission can occur directly between child-caregiver dyads as well as through other pathways involving water and livestock. Additional research into caregivers as a source of childhood infection is warranted.
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http://dx.doi.org/10.1093/ofid/ofaa533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7731525PMC
December 2020

Reduced treatment frequencies with bumped kinase inhibitor 1369 are effective against porcine cystoisosporosis.

Int J Parasitol Drugs Drug Resist 2020 12 21;14:37-45. Epub 2020 Aug 21.

Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine, Veterinärplatz 1, A-1210, Vienna, Austria.

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.
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http://dx.doi.org/10.1016/j.ijpddr.2020.08.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7442133PMC
December 2020

Conducting clinical trials in sub-Saharan Africa: challenges and lessons learned from the Malawi Cryptosporidium study.

Trials 2020 Jul 25;21(1):680. Epub 2020 Jul 25.

Malawi-Liverpool Wellcome Trust Clinical Research Programme, Blantyre, Malawi.

Background: An effective drug to treat cryptosporidial diarrhea in HIV-infected individuals is a global health priority. Promising drugs need to be evaluated in endemic areas which may be challenged by both lack of resources and experience to conduct International Committee of Harmonisation-Good Clinical Practice (ICH-GCP)-compliant clinical trials.

Methods: We present the challenges and lessons learned in implementing a phase 2A, randomized, double-blind, placebo-controlled trial of clofazimine, in treatment of cryptosporidiosis among HIV-infected adults at a single site in Malawi.

Results: Primary challenges are grouped under study initiation, study population, study implementation, and cultural issues. The lessons learned primarily deal with regulatory system and operational barriers, and recommendations can be applied to other human experimental trials in low- and middle-income countries, specifically in sub-Saharan Africa.

Conclusion: This study demonstrated that initiating and implementing human experimental trials in sub-Saharan Africa can be challenging. However, solutions exist and successful execution requires careful planning, ongoing evaluation, responsiveness to new developments, and oversight of all trial operations.
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http://dx.doi.org/10.1186/s13063-020-04620-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382797PMC
July 2020

Comparative assessment of the effects of bumped kinase inhibitors on early zebrafish embryo development and pregnancy in mice.

Int J Antimicrob Agents 2020 Sep 21;56(3):106099. Epub 2020 Jul 21.

Institute of Parasitology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Länggass-Strasse 122, CH-3012 Bern, Switzerland. Electronic address:

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 were applied by gavage for 5 days. Five BKIs did not affect pregnancy, five BKIs exhibited ~15-35% neonatal mortality and five 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 fertilised eggs to 0.2-50 μM of BKIs and microscopic monitoring of embryo development in a blinded manner for 4 days. We propose an algorithm that includes quantification of malformations and embryo deaths, and established a scoring system that allows the calculation of an impact score (S) indicating at which concentrations BKIs visibly affect zebrafish embryo development. Comparison of the two models showed that for nine compounds no clear correlation between S and pregnancy outcome was observed. However, the three BKIs affecting zebrafish embryos only at high concentrations (≥40 μM) did not impair mouse pregnancy at all, and the three compounds that inhibited zebrafish embryo development already at 0.2 μM showed detrimental effects in the pregnancy model. Thus, the zebrafish embryo development test has 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.
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http://dx.doi.org/10.1016/j.ijantimicag.2020.106099DOI Listing
September 2020

Taming the Boys for Global Good: Contraceptive Strategy to Stop Malaria Transmission.

Molecules 2020 Jun 16;25(12). Epub 2020 Jun 16.

Center for Emerging and Re-emerging Infectious Diseases (CERID), Division of Allergy and Infectious Diseases, Department of Medicine, University of Washington, Seattle, WA 98109, USA.

Transmission of human malaria parasites ( spp.) by 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 transmission from humans to mosquitoes with novel transmission-blocking remedies would be an important complement to other interventions in reducing malaria incidence.
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http://dx.doi.org/10.3390/molecules25122773DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7356879PMC
June 2020

: Differential Proteome of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294.

Microorganisms 2020 May 26;8(6). Epub 2020 May 26.

Institute of Parasitology, Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Länggass-Strasse 122, 3012 Bern, Switzerland.

Background: the apicomplexan parasite causes important reproductive problems in farm animals, most notably in cattle. After infection via oocysts or tissue cysts, rapidly dividing tachyzoites infect various tissues and organs, and in immunocompetent hosts, they differentiate into slowly dividing bradyzoites, which form tissue cysts and constitute a resting stage persisting within infected tissues. Bumped kinase inhibitors (BKIs) of calcium dependent protein kinase 1 are promising drug candidates for the treatment of infections. BKI-1294 exposure of cell cultures infected with tachyzoites results in the formation of massive multinucleated complexes (MNCs) containing numerous newly formed zoites, which remain viable for extended periods of time under drug pressure in vitro. MNC and tachyzoites exhibit considerable antigenic and structural differences.

Methods: Using shotgun mass spectrometry, we compared the proteomes of tachyzoites to BKI-1294 induced MNCs, and analyzed the mRNA expression levels of selected genes in both stages.

Results: More than half of the identified proteins are downregulated in MNCs as compared to tachyzoites. Only 12 proteins are upregulated, the majority of them containing SAG1 related sequence (SRS) domains, and some also known to be expressed in bradyzoites Conclusions: MNCs exhibit a proteome different from tachyzoites, share some bradyzoite-like features, but may constitute a third stage, which remains viable and ensures survival under adverse conditions such as drug pressure. We propose the term "baryzoites" for this stage (from Greek βαρυσ = massive, bulky, heavy, inert).
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http://dx.doi.org/10.3390/microorganisms8060801DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355844PMC
May 2020

: Structure and Fate of Multinucleated Complexes Induced by the Bumped Kinase Inhibitor BKI-1294.

Pathogens 2020 May 16;9(5). Epub 2020 May 16.

Institute of Parasitology, Vetsuisse Faculty, University of Bern, 3012 Bern, Switzerland.

Background: Bumped kinase inhibitors (BKIs) are potential drugs for neosporosis treatment in farm animals. BKI-1294 exposure results in the formation of multinucleated complexes (MNCs), which remain viable in vitro under constant drug pressure. We investigated the formation of BKI-1294 induced MNCs, the re-emergence of viable tachyzoites following drug removal, and the localization of CDPK1, the molecular target of BKIs.

Methods: tachyzoites and MNCs were studied by TEM and immunofluorescence using antibodies directed against CDPK1, and against NcSAG1 and IMC1 as markers for tachyzoites and newly formed zoites, respectively.

Results: After six days of drug exposure, MNCs lacked SAG1 surface expression but remained intracellular, and formed numerous zoites incapable of disjoining from each other. Following drug removal, proliferation continued, and zoites lacking NcSAG1 emerged from the periphery of these complexes, forming infective tachyzoites after 10 days. In intracellular tachyzoites, CDPK1 was evenly distributed but shifted towards the apical part once parasites were extracellular. This shift was not affected by BKI-1294.

Conclusions: CDPK1 has a dynamic distribution depending on whether parasites are located within a host cell or outside. During MNC-to-tachyzoite reconversion newly formed tachyzoites are generated directly from MNCs through zoites of unknown surface antigen composition. Further in vivo studies are needed to determine if MNCs could lead to a persistent reservoir of infection after BKI treatment.
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http://dx.doi.org/10.3390/pathogens9050382DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281336PMC
May 2020

Bumped Kinase Inhibitors as therapy for apicomplexan parasitic diseases: lessons learned.

Int J Parasitol 2020 05 26;50(5):413-422. Epub 2020 Mar 26.

Department of Medicine, Division of Allergy and Infectious Diseases, Center for Emerging and Reemerging Infectious Diseases (CERID), University of Washington, Seattle, WA 98109, USA. Electronic address:

Bumped Kinase Inhibitors, targeting Calcium-dependent Protein Kinase 1 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.
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http://dx.doi.org/10.1016/j.ijpara.2020.01.006DOI Listing
May 2020

Toward a structome of Acinetobacter baumannii drug targets.

Protein Sci 2020 03 20;29(3):789-802. Epub 2020 Jan 20.

Department of Medicine, Division of Allergy and Infectious Disease, Center for Emerging and Re-emerging Infectious Disease (CERID), University of Washington, Seattle, Washington.

Acinetobacter baumannii is well known for causing hospital-associated infections due in part to its intrinsic antibiotic resistance as well as its ability to remain viable on surfaces and resist cleaning agents. In a previous publication, A. baumannii strain AB5075 was studied by transposon mutagenesis and 438 essential gene candidates for growth on rich-medium were identified. The Seattle Structural Genomics Center for Infectious Disease entered 342 of these candidate essential genes into our pipeline for structure determination, in which 306 were successfully cloned into expression vectors, 192 were detectably expressed, 165 screened as soluble, 121 were purified, 52 crystalized, 30 provided diffraction data, and 29 structures were deposited in the Protein Data Bank. Here, we report these structures, compare them with human orthologs where applicable, and discuss their potential as drug targets for antibiotic development against A. baumannii.
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http://dx.doi.org/10.1002/pro.3826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020997PMC
March 2020

Structures of glyceraldehyde 3-phosphate dehydrogenase in Neisseria gonorrhoeae and Chlamydia trachomatis.

Protein Sci 2020 03 28;29(3):768-778. Epub 2020 Jan 28.

Department of Medicine, Division of Allergy and Infectious Diseases, Center for Emerging and Re-emerging Infectious Diseases (CERID), University of Washington, Seattle, Washington.

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.
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http://dx.doi.org/10.1002/pro.3824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020975PMC
March 2020

Solution structure for an Encephalitozoon cuniculi adrenodoxin-like protein in the oxidized state.

Protein Sci 2020 03 20;29(3):809-817. Epub 2020 Jan 20.

Seattle Structural Genomics Center for Infectious Diseases, Seattle, Washington.

Encephalitozoon cuniculi is a unicellular, obligate intracellular eukaryotic parasite in the Microsporidia family and one of the agents responsible for microsporidosis infections in humans. Like most Microsporidia, the genome of E. cuniculi is markedly reduced and the organism contains mitochondria-like organelles called mitosomes instead of mitochondria. Here we report the solution NMR structure for a protein physically associated with mitosome-like organelles in E. cuniculi, the 128-residue, adrenodoxin-like protein Ec-Adx (UniProt ID Q8SV19) in the [2Fe-2S] ferredoxin superfamily. Oxidized Ec-Adx contains a mixed four-strand β-sheet, β2-β1-β4-β3 (↓↑↑↓), loosely encircled by three α-helices and two 3 -helices. This fold is similar to the structure observed in other adrenodoxin and adrenodoxin-like proteins except for the absence of a fifth anti-parallel β-strand next to β3 and the position of α3. Cross peaks are missing or cannot be unambiguously assigned for 20 amide resonances in the H- N HSQC spectrum of Ec-Adx. These missing residues are clustered primarily in two regions, G48-V61 and L94-L98, containing the four cysteine residues predicted to ligate the paramagnetic [2Fe-2S] cluster. Missing amide resonances in H- N HSQC spectra are detrimental to NMR-based solution structure calculations because H- H NOE restraints are absent (glass half-empty) and this may account for the absent β-strand (β5) and the position of α3 in oxidized Ec-Adx. On the other hand, the missing amide resonances unambiguously identify the presence, and immediate environment, of the paramagnetic [2Fe-2S] cluster in oxidized Ec-Adx (glass half-full).
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http://dx.doi.org/10.1002/pro.3818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7020980PMC
March 2020

Special Issue Focused on Gut Pathogens.

ACS Infect Dis 2020 01 23;6(1):1-2. Epub 2019 Dec 23.

Global Health Pharma , GlaxoSmithKline , Tres Cantos 28760 , Spain.

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http://dx.doi.org/10.1021/acsinfecdis.9b00432DOI Listing
January 2020

Chemoprophylaxis Vaccination: Phase I Study to Explore Stage-specific Immunity to Plasmodium falciparum in US Adults.

Clin Infect Dis 2020 09;71(6):1481-1490

Center for Infectious Disease Research, Seattle, Washington, USA.

Background: Chemoprophylaxis vaccination with sporozoites (CVac) with chloroquine induces protection against a homologous Plasmodium falciparum sporozoite (PfSPZ) challenge, but whether blood-stage parasite exposure is required for protection remains unclear. Chloroquine suppresses and clears blood-stage parasitemia, while other antimalarial drugs, such as primaquine, act against liver-stage parasites. Here, we evaluated CVac regimens using primaquine and/or chloroquine as the partner drug to discern whether blood-stage parasite exposure impacts protection against homologous controlled human malaria infection.

Methods: In a Phase I, randomized, partial double-blind, placebo-controlled study of 36 malaria-naive adults, all CVac subjects received chloroquine prophylaxis and bites from 12-15 P. falciparum-infected mosquitoes (CVac-chloroquine arm) at 3 monthly iterations, and some received postexposure primaquine (CVac-primaquine/chloroquine arm). Drug control subjects received primaquine, chloroquine, and uninfected mosquito bites. After a chloroquine washout, subjects, including treatment-naive infectivity controls, underwent homologous, PfSPZ controlled human malaria infection and were monitored for parasitemia for 21 days.

Results: No serious adverse events occurred. During CVac, all but 1 subject in the study remained blood-smear negative, while only 1 subject (primaquine/chloroquine arm) remained polymerase chain reaction-negative. Upon challenge, compared to infectivity controls, 3/3 chloroquine arm subjects displayed delayed patent parasitemia (P = .01) but not sterile protection, while 3/11 primaquine/chloroquine subjects remained blood-smear negative.

Conclusions: CVac-primaquine/chloroquine is safe and induces sterile immunity to P. falciparum in some recipients, but a single 45 mg dose of primaquine postexposure does not completely prevent blood-stage parasitemia. Unlike previous studies, CVac-chloroquine did not produce sterile immunity.

Clinical Trials Registration: NCT01500980.
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http://dx.doi.org/10.1093/cid/ciz1010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7486848PMC
September 2020

P-Glycoprotein-Mediated Efflux Reduces the In Vivo Efficacy of a Therapeutic Targeting the Gastrointestinal Parasite Cryptosporidium.

J Infect Dis 2019 08;220(7):1188-1198

Division of Allergy and Infectious Disease, Department of Medicine, University of Washington, Seattle.

Recent studies have illustrated the burden Cryptosporidium infection places on the lives of malnourished children and immunocompromised individuals. Treatment options remain limited, and efforts to develop a new therapeutic are currently underway. However, there are unresolved questions about the ideal pharmacokinetic characteristics of new anti-Cryptosporidium therapeutics. Specifically, should drug developers optimize therapeutics and formulations to increase drug exposure in the gastrointestinal lumen, enterocytes, or systemic circulation? Furthermore, how should researchers interpret data suggesting their therapeutic is a drug efflux transporter substrate? In vivo drug transporter-mediated alterations in efficacy are well recognized in multiple disease areas, but the impact of intestinal transporters on therapeutic efficacy against enteric diseases has not been established. Using multiple in vitro models and a mouse model of Cryptosporidium infection, we characterized the effect of P-glycoprotein efflux on bumped kinase inhibitor pharmacokinetics and efficacy. Our results demonstrated P-glycoprotein decreases bumped kinase inhibitor enterocyte exposure, resulting in reduced in vivo efficacy against Cryptosporidium. Furthermore, a hollow fiber model of Cryptosporidium infection replicated the in vivo impact of P-glycoprotein on anti-Cryptosporidium efficacy. In conclusion, when optimizing drug candidates targeting the gastrointestinal epithelium or gastrointestinal epithelial infections, drug developers should consider the adverse impact of active efflux transporters on efficacy.
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http://dx.doi.org/10.1093/infdis/jiz269DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6736360PMC
August 2019

Treatment with Bumped Kinase Inhibitor 1294 Is Safe and Leads to Significant Protection against Abortion and Vertical Transmission in Sheep Experimentally Infected with Toxoplasma gondii during Pregnancy.

Antimicrob Agents Chemother 2019 07 24;63(7). Epub 2019 Jun 24.

SALUVET, Animal Health Department, Faculty of Veterinary Sciences, Complutense University of Madrid, Madrid, Spain

Previous studies on drug efficacy showed low protection against abortion and vertical transmission of in pregnant sheep. 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 and in laboratory animal models. Here, we present the safety and efficacy of BKI-1294 treatment (dosed orally at 100 mg/kg of body weight 5 times every 48 h) initiated 48 h after oral infection of sheep at midpregnancy with 1,000 TgShSp1 oocysts. BKI-1294 demonstrated systemic exposure in pregnant ewes, with maximum plasma concentrations of 2 to 3 μM and trough concentrations of 0.4 μM at 48 h after each dose. Oral administration of BKI-1294 in uninfected sheep at midpregnancy was deemed safe, since there were no changes in behavior, fecal consistency, rectal temperatures, hematological and biochemical parameters, or fetal mortality/morbidity. In ewes infected with a oocyst dose lethal for fetuses, BKI-1294 treatment led to a minor rectal temperature increase after infection and a decrease in fetal/lamb mortality of 71%. None of the lambs born alive in the treated group exhibited congenital encephalitis lesions, and vertical transmission was prevented in 53% of them. BKI-1294 treatment during infection led to strong interferon gamma production after cell stimulation and a low humoral immune response to soluble tachyzoite antigens but high levels of anti-SAG1 antibodies. The results demonstrate a proof of concept for the therapeutic use of BKI-1294 to protect ovine fetuses from infection during pregnancy.
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http://dx.doi.org/10.1128/AAC.02527-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6591617PMC
July 2019

Bumped kinase inhibitor 1369 is effective against Cystoisospora suis in vivo and in vitro.

Int J Parasitol Drugs Drug Resist 2019 08 2;10:9-19. Epub 2019 Apr 2.

Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine, Veterinärplatz 1, A-1210, Vienna, Austria.

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.
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http://dx.doi.org/10.1016/j.ijpddr.2019.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6453670PMC
August 2019

Lysyl-tRNA synthetase as a drug target in malaria and cryptosporidiosis.

Proc Natl Acad Sci U S A 2019 04 20;116(14):7015-7020. Epub 2019 Mar 20.

Department of Life Sciences, Imperial College, South Kensington, SW7 2AZ London, United Kingdom.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage and in cell-culture studies. Target deconvolution in has shown that cladosporin inhibits lysyl-tRNA synthetase (KRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both KRS1 and KRS (KRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between KRS1 and KRS. This series of compounds inhibit KRS and and in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for KRS1 and KRS vs. (human) KRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis.
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http://dx.doi.org/10.1073/pnas.1814685116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6452685PMC
April 2019

Development of 5-Aminopyrazole-4-carboxamide-based Bumped-Kinase Inhibitors for Cryptosporidiosis Therapy.

J Med Chem 2019 03 15;62(6):3135-3146. Epub 2019 Mar 15.

Department of Medicine, Division of Allergy & Infectious Disease, Center for Emerging & Re-Emerging Infectious Disease (CERID) , University of Washington , Seattle , Washington 98109 , United States.

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 preclinical leads for cryptosporidiosis therapy with acceptable safety parameters and efficacy in the mouse model of cryptosporidiosis.
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http://dx.doi.org/10.1021/acs.jmedchem.9b00069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6559944PMC
March 2019

Enzymatic and Structural Characterization of the Glucokinase.

Antimicrob Agents Chemother 2019 05 25;63(5). Epub 2019 Apr 25.

Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson University, Clemson, South Carolina, USA

Infection with the free-living amoeba leads to life-threatening primary amoebic meningoencephalitis. Efficacious treatment options for these infections are limited, and the mortality rate is very high (∼98%). Parasite metabolism may provide suitable targets for therapeutic design. Like most other organisms, glucose metabolism is critical for parasite viability, being required for growth in culture. The first enzyme required for glucose metabolism is typically a hexokinase (HK), which transfers a phosphate from ATP to glucose. The products of this enzyme are required for both glycolysis and the pentose phosphate pathway. However, the genome lacks an obvious HK homolog and instead harbors a glucokinase (Glck). The Glck (NfGlck) shares limited (25%) amino acid identity with the mammalian host enzyme ( Glck), suggesting that parasite-specific inhibitors with anti-amoeba activity can be generated. Following heterologous expression, NfGlck was found to have a limited hexose substrate range, with the greatest activity observed with glucose. The enzyme had apparent values of 42.5 ± 7.3 μM and 141.6 ± 9.9 μM for glucose and ATP, respectively. The NfGlck structure was determined and refined to 2.2-Å resolution, revealing that the enzyme shares greatest structural similarity with the Glck. These similarities include binding modes and binding environments for substrates. To identify inhibitors of NfGlck, we screened a small collection of inhibitors of glucose-phosphorylating enzymes and identified several small molecules with 50% inhibitory concentration values of <1 μM that may prove useful as hit chemotypes for further leads and therapeutic development against .
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http://dx.doi.org/10.1128/AAC.02410-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6496100PMC
May 2019