Background Methionyl-tRNA synthetase (MetRS) inhibitors are under investigation for the treatment of intestinal infections caused by Giardia lamblia. Objectives To properly analyse the therapeutic potential of the MetRS inhibitor 1717, experimental tools including a robust cell-based assay and a murine model of infection were developed based on novel strains of G. lamblia that employ luciferase reporter systems to quantify viable parasites. Methods Systematic screening of Giardia-specific promoters and luciferase variants led to the development of a strain expressing the click beetle green luciferase. Further modifying this strain to express NanoLuc created a dual reporter strain capable of quantifying parasites in both the trophozoite and cyst stages. These strains were used to develop a high-throughput cell assay and a mouse infection model. A library of MetRS inhibitors was screened in the cell assay and Compound-1717 was tested for efficacy in the mouse infection model. Results Cell viability in in vitro compound screens was quantified via bioluminescence readouts while infection loads in mice were monitored with non-invasive whole-animal imaging and faecal analysis. Compound-1717 was effective in clearing mice of Giardia infection in 3 days at varying doses, which was supported by data from enzymatic and phenotypic cell assays. Conclusions The new in vitro and in vivo assays based on luciferase expression by engineered G. lamblia strains are useful for the discovery and development of new therapeutics for giardiasis. MetRS inhibitors, as validated by Compound-1717, have promising anti-giardiasis properties that merit further study as alternative 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 ( Cs CDPK1) was identified and cloned to investigate activity and thermal stabilization of the recombinant Cs CDPK1 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.

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.

G protein-coupled receptor (GPCR) biogenesis, trafficking, and function are regulated by posttranslational modifications, including N-glycosylation of asparagine residues. α 1D-adrenergic receptors (α 1D-ARs)-key regulators of central and autonomic nervous system function-contain two putative N-glycosylation sites within the large N-terminal domain at N65 and N82. However, determining the glycosylation state of this receptor has proven challenging. Towards understanding the role of these putative glycosylation sites, site-directed mutagenesis and lectin affinity purification identified N65 and N82 as bona fide acceptors for N-glycans. Surprisingly, we also report that simultaneously mutating N65 and N82 causes early termination of α 1D-AR between transmembrane domain 2 and 3. Labelfree dynamic mass redistribution and cell surface trafficking assays revealed that single and double glycosylation deficient mutants display limited function with impaired plasma membrane expression. Confocal microscopy imaging analysis and SNAP-tag sucrose density fractionation assays revealed the dual glycosylation mutant α 1D-AR is widely distributed throughout the cytosol and nucleus. Based on these novel findings, we propose α 1D-AR transmembrane domain 2 acts as an ER localization signal during active protein biogenesis, and that α 1D-AR n-terminal glycosylation is required for complete translation of nascent, functional receptor. G protein-coupled receptors (GPCRs) are essential membrane proteins that regulate the vast majority of physiological functions in the human body. As a result, GPCRs have been estimated to be targeted by approximately one third of all currently approved medications 1. Adrenergic receptors (ARs) are a clinically relevant subfamily of GPCRs. Activated by the endogenous sympathetic neurotransmitters epinephrine and norepinephrine, adrenergic GPCRs consist of three major subtypes: α 1 , α 2 , and β. The α 1 sub-family-containing α 1A , α 1B , and α 1D subtypes 2-are targets for medications that regulate blood pressure 3,4 , bladder 5,6 , prostate 7,8 , and central nervous system function 9-11. Thus, understanding the molecular and cellular mechanisms regulating α 1-AR function will help spur the development of new medications associated with aberrant α 1-AR signaling, such as hypertension, PTSD, schizophrenia, and benign prostatic hypertrophy 12-15. Among the three α 1 subtypes, the α 1D-AR remains poorly understood due to technical challenges. Relative to the closely related α 1A and α 1B-AR subtypes, α 1D-AR displays limited functional responses and minimal plasma membrane expression when expressed in heterologous cell culture 16-18. Although pharmacologically detectable in intact isolated aortae in organ-tissue bath assays 19 , α 1D-AR functional expression rapidly disappears in primary vascular smooth muscle cell cultures within 24-48 hours 20. Also, immortalized cell lines that endogenously express α 1D-ARs have yet to be discovered. Combined, these experimental clues indicate the molecular and cellular ...

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