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We have developed compounds with a promising activity against Acinetobacter baumannii and Pseudomonas aeruginosa, which are both on the WHO priority list of antibiotic-resistant bacteria. Starting from DNA gyrase inhibitor 1, we identified compound 27, featuring a 10-fold improved aqueous solubility, a 10-fold improved inhibition of topoisomerase IV from A. baumannii and P. aeruginosa, a 10-fold decreased inhibition of human topoisomerase IIα, and no cross-resistance to novobiocin. Cocrystal structures of 1 in complex with Escherichia coli GyrB24 and (S)-27 in complex with A. baumannii GyrB23 and P. aeruginosa GyrB24 revealed their binding to the ATP-binding pocket of the GyrB subunit. In further optimization steps, solubility, plasma free fraction, and other ADME properties of 27 were improved by fine-tuning of lipophilicity. In particular, analogs of 27 with retained anti-Gram-negative activity and improved plasma free fraction were identified. The series was found to be nongenotoxic, nonmutagenic, devoid of mitochondrial toxicity, and possessed no ion channel liabilities. Opis vira z dne 19. 1. 2023. Nasl. z nasl. zaslona. Bibliografija: str. 1423-1425. Abstract. ARRS, Farmacevtska kemija: načrtovanje, sinteza in vrednotenje učinkovin ARRS, MTAvsAMR: novi večtarčni antibiotiki proti večkratno odpornim bakterijam ARRS, Razvoj novih zaviralcev bakterijskih topoizomeraz za boj proti odpornim infekcijam iCASE studentship funded by BBSRC and Redx Pharma Plc Investigator Award from the Wellcome Trust BBSRC Institute Strategic Programme

A new series of dual low nanomolar benzothiazole inhibitors of bacterial DNA gyrase and topoisomerase IV were developed. The resulting compounds show excellent broad-spectrum antibacterial activities against Gram-positive Enterococcus faecalis, Enterococcus faecium and multidrug resistant (MDR) Staphylococcus aureus strains [best compound minimal inhibitory concentrations (MICs): range

Peptide drugs and biologics provide opportunities for treatments of many diseases. However, due to their poor stability and permeability in the gastrointestinal tract, the oral bioavailability of peptide drugs is negligible. Nanoparticle formulations have been proposed to circumvent these hurdles, but systemic exposure of orally administered peptide drugs has remained elusive. In this study, we investigated the absorption mechanisms of four insulin-loaded arginine-rich nanoparticles displaying differing composition and surface characteristics, developed within the pan-European consortium TRANS-INT. The transport mechanisms and major barriers to nanoparticle permeability were investigated in freshly isolated human jejunal tissue. Cytokine release profiles and standard toxicity markers indicated that the nanoparticles were nontoxic. Three out of four nanoparticles displayed pronounced binding to the mucus layer and did not reach the epithelium. One nanoparticle composed of a mucus inert shell and cell-penetrating octarginine (ENCP), showed significant uptake by the intestinal epithelium corresponding to 28 +/- 9% of the administered nanoparticle dose, as determined by super-resolution microscopy. Only a small fraction of nanoparticles taken up by epithelia went on to be transcytosed via a dynamin-dependent process. In situ studies in intact rat jejunal loops confirmed the results from human tissue regarding mucus binding, epithelial uptake, and negligible insulin bioavailability. In conclusion, while none of the four arginine-rich nanoparticles supported systemic insulin delivery, ENCP displayed a consistently high uptake along the intestinal villi. It is proposed that ENCP should be further investigated for local delivery of therapeutics to the intestinal mucosa. Funding Agencies|European TRANS-INT Consortium; European Union [281035]; TRANS-INT; Swedish Research Council [2822, 2020-01586]; FRS-FNRS (Fonds de la Recherche Scientifique) , Belgium; Competitive Reference Groups, Conselleria de Educacion e Ordenacion Universitaria, Xunta de Galicia [ED431C 2021/17]; European Commission, Education, Audiovisual and Culture Executive Agency (EACEA) [20120028]; Swedish Government, Sweden

Acamprosate is an anionic drug substance widely used in treating symptoms of alcohol withdrawal. It was recently shown that oral acamprosate absorption is likely due to paracellular transport. In contrast, little is known about the eliminating mechanism clearing acamprosate from the blood in the kidneys, despite the fact that studies have shown renal secretion of acamprosate. The hypothesis of the present study was therefore that renal organic anion transporters (OATs) facilitate the renal excretion of acamprosate in humans. The aim of the present study was to establish and apply OAT1 (gene product of SLC22A6) and OAT3 (gene product of SLC22A8) expressing cell lines to investigate whether acamprosate is a substrate or inhibitor of OAT1 and/or OAT3. The studies were performed in HEK293-Flp-In cells stably transfected with SLC22A6 or SLC22A8. Protein and functional data showed that the established cell lines are useful for studying OAT1- and OAT3-mediated transport in bi-laboratory studies. Acamprosate inhibited OAT1-mediated p-aminohippuric acid (PAH) uptake but did not inhibit substrate uptake via OAT3 expressing cells, neither when applied concomitantly nor after a 3 h preincubation with acamprosate. The uptake of PAH via OAT1 was inhibited in a competitive manner by acamprosate and cellular uptake studies showed that acamprosate is a substrate for OAT1 with a Km-value of approximately 700 &micro, M. Probenecid inhibited OAT1-mediated acamprosate uptake with a Ki-value of approximately 13 &micro, M, which may translate into an estimated clinically significant DDI index. In conclusion, acamprosate was identified as a substrate of OAT1 but not OAT3.

Madin-Darby canine kidney (MDCK) II cells stably transfected with transport proteins are commonly used models for drug transport studies. However, endogenous expression of especially canine MDR1 (cMDR1) confounds the interpretation of such studies. Here we have established an MDCK cell line stably overexpressing the human MDR1 transporter (hMDR1; P-glycoprotein), and used CRISPR-Cas9 gene editing to knockout the endogenous cMDR1. Genomic screening revealed the generation of a clonal cell line homozygous for a 4-nucleotide deletion in the canine ABCB1 gene leading to a frameshift and a premature stop codon. Knockout of cMDR1 expression was verified by quantitative protein analysis and functional studies showing retained activity of the human MDR1 transporter. Application of this cell line allowed unbiased reclassification of drugs previously defined as both substrates and non-substrates in different studies using commonly used MDCK-MDR1 clones. Our new MDCK-hMDR1 cell line, together with a previously developed control cell line, both with identical deletions in the canine ABCB1 gene and lack of cMDR1 expression represent excellent in vitro tools for use in drug discovery.

Introduction. Acamprosate is a BCS class III, non-metabolized, anionic drug substance for which the excretion mechanism is not fully understood. Intravenous administration in rats of acamprosate together with probenecid, a known substrate and inhibitor of the organic anion transporter (OAT) 1 (SLC22A6) (1), decreased the renal clearance (ClR) of acamprosate in a dose-dependent manner (2). The hypothesis of the present study was that renal OATs expressed in the basolateral membrane of the proximal tubule epithelium (3) could be the carrier contributing to renal excretion of acamprosate and the reason for potential acamprosate-drug (e.g. probenecid) interactions.Aim. To investigate if acamprosate interacts with OAT1 by measuring the concentration-dependent effect of acamprosate on the uptake of [14C]-p-aminohippuric acid ([14C]-PAH), a well-established substrate of OAT1 (4).Method. The apical uptake of 0.5 μCi/mL [14C]-PAH was measured for 5 minutes at 37°C and 100 rpm in the presence of 0 – 33 000 μM acamprosate in human embryonic kidney (HEK)293 cells transiently expressing OAT1 (Corning TransportoCells OAT1), or in a HEK293-Flp-in cell line stably expressing OAT1 (developed in-house). The uptake was in both series measured in parallel in mock-transfected HEK293 cells grown under similar cell culture conditions.Results. A significant, time-dependent and saturable increase of [14C]-PAH apical uptake was observed in the OAT1-transfected HEK293 cells compared to mock-transfected cells. The apical uptake of [14C]-PAH in OAT1-transfected cells was decreased in the presence of acamprosate in a concentration-dependent manner, while no effect of acamprosate was observed in the mock-transfected cells. The mean (+/- SD) inhibition constant for [14C]-PAH uptake (IC50) was 1 042 ± 134 and 902 ± 20 μM in the transiently (n=3) and stably transfected (N=3, n=2) HEK293-OAT1 cells, respectively.Conclusion. Acamprosate inhibits OAT1 in vitro at concentrations close to the maximum unbound plasma concentration of acamprosate, cmax = 154-768 μM (5), obtained after intravenous administration [333-2130 mg].References1. Nigam SK, Bush KT, Martovetsky G, Ahn SY, Liu HC, Richard E, et al. The organic anion transporter (OAT) family: a systems biology perspective. Physiol Rev. 2015;95(1):83-123.2. Zornoza T, Guerri C, Polache A, Granero L. Disposition of acamprosate in the rat: influence of probenecid. Biopharm Drug Dispos. 2002;23(7):283-91.3. Wright SH, Dantzler WH. Molecular and cellular physiology of renal organic cation and anion transport. Physiol Rev. 2004;84(3):987-1049.4. FDA. In Vitro Metabolism and Transporter-Mediated Drug-Drug Interaction Studies - Guidance for Industry. In: CDER, editor. Silver Spring, MD: U.S. Department of Health and Human Services; 2017. p. 1-37.5. Approval package for application number 21-431: Campral clinical pharmacology and biopharmaceutics review: Center for drug evaluation and research; 2004 [cited 2018 08 13]. Available from: https://www.accessdata.fda.gov/drugsatfda_docs/nda/2004/21-431_Campral_BioPharmr.pdf.

Madin-Darby canine kidney II cells transfected with one or several transport proteins are commonly used models to study drug transport. In these cells, however, endogenous transporters such as canine Mdr1/P-glycoprotein (Abcb1) complicate the interpretation of transport studies. The aim of this investigation was to establish a Madin-Darby canine kidney II cell line using CRISPR-Cas9 gene-editing technology to knock out endogenous canine Mdr1 (cMdr1) expression. CRISPR-Cas9-mediated Abcb1 homozygous disruption occurred at frequencies of around 20% and resulted in several genotypes. We selected 1 clonal cell line, cMdr1 KO Cl2, for further examination. Consistent with an on-target effect of CRISPR-Cas9 in specific regions of the endogenous canine Abcb1 gene, we obtained a cell clone with Abcb1 gene alterations and without any cMdr1 expression, as confirmed by genome sequencing and quantitative protein analysis. Functional studies of these cells, using digoxin and other prototypic MDR1 substrates, showed close to identical transport in the apical-to-basolateral and basolateral-to-apical directions, resulting in efflux ratios indistinguishable from unity.

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9), i.e., CRISPR-Cas9, has been extensively used as a gene-editing technology during recent years. Unlike earlier technologies for gene editing or gene knockdown, such as zinc finger nucleases and RNA interference, CRISPR-Cas9 is comparably easy to use, affordable, and versatile. Recently, CRISPR-Cas9 has been applied in studies of drug absorption, distribution, metabolism, and excretion (ADME) and for ADME model generation. To date, about 50 papers have been published describing in vitro or in vivo CRISPR-Cas9 gene editing of ADME and ADME-related genes. Twenty of these papers describe gene editing of clinically relevant genes, such as ATP-binding cassette drug transporters and cytochrome P450 drug-metabolizing enzymes. With CRISPR-Cas9, the ADME tool box has been substantially expanded. This new technology allows us to develop better and more predictive in vitro and in vivo ADME models and map previously underexplored ADME genes and gene families. In this mini-review, we give an overview of the CRISPR-Cas9 technology and summarize recent applications of CRISPR-Cas9 within the ADME field. We also speculate about future applications of CRISPR-Cas9 in ADME research.

The aim of this study was to analyze the functional importance of the C-terminus of the essential yeast ribosomal protein L5 (YrpL5). Previous studies have indicated that the C-terminal region of YrpL5 forms an alpha-helix with a positively charged surface that is involved in protein-5S rRNA interaction. Formation of an YrpL5.5S rRNA complex is a prerequisite for nuclear import of YrpL5. Here we have tested the importance of the alpha-helix and the positively charged surface for YrpL5 function in Saccharomyces cerevisiae using site directed mutagenesis in combination with functional complementation. Alterations in the sequence forming the putative alpha-helix affected the functional capacity of YrpL5. However, the effect did not correlate with a decreased ability of the protein to bind to 5S rRNA as all rpL5 mutants tested were imported to the nucleus whether or not the alpha-helix or the positively charged surface were intact. The alterations introduced in the C-terminal sequence affected the growth rate of cells expressing mutant but functional forms of YrpL5. The reduced growth rate was correlated with a reduced ribosomal content per cell indicating that the alterations introduced in the C-terminus interfered with ribosome assembly.