Benzylic Dehydroxylation of Echinocandin antifungal drugs restores efficacy against resistance conferred by mutated Glucan Synthase

Each year, infections caused by fungal pathogens claim the lives of about 1.6 million people and affect the health of over a billion people worldwide. Among the most recently developed antifungal drugs are the echinocandins, which noncompetitively inhibit β-glucan synthase, a membrane-bound protein complex that catalyzes the formation of the main polysaccharide component of the fungal cell wall. Resistance to echinocandins is conferred by mutations in FKS genes, which encode the catalytic subunit of the β-glucan synthase complex. Here, we report that selective removal of the benzylic alcohol of the nonproteinogenic amino acid 3S,4S-dihydroxy-l-homotyrosine of the echinocandins anidulafungin and rezafungin, restored their efficacy against a large panel of echinocandin-resistant Candida strains. The dehydroxylated compounds did not significantly affect the viability of human-derived cell culture lines. An analysis of the efficacy of the dehydroxylated echinocandins against resistant Candida strains, which contain mutations in the FKS1 and/or FKS2 genes of the parental strains, identified amino acids of the Fks proteins that are likely to reside in proximity to the l-homotyrosine residue of the bound drug. This study describes the first example of a chemical modification strategy to restore the efficacy of echinocandin drugs, which have a critical place in the arsenal of antifungal drugs, against resistant fungal pathogens.The authors thank J. Berman and D. Perlin for providing Candida strains. This work was supported by the Israel Science Foundation Grant 179/19 (Micha Fridman). TG acknowledges support from the Spanish Ministry of Science and Innovation for grant PGC2018-099921-B-I00 and from the “la Caixa” Foundation under the agreements LCF/PR/GN18/50310010 and LCF/PR/HR21/00737. They also thank E. Ainbinder. O. Singer, and Y. Fried from the Stem Cell Unit of Life Science Core Facilities, the Weizmann Institute of Science. They especially thank H. Barr, head of HTS and Medicinal Chemistry Units at the Maurice and Vivienne Wohl Institute for Drug Discovery, The Nancy and Stephen Grand Israel National Center for Personalized Medicine, Weizmann Institute of Science.Peer ReviewedPostprint (published version

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Elevated vacuolar uptake of fluorescently labeled antifungal drug caspofungin predicts echinocandin resistance in pathogenic yeast

Echinocandins are the newest class of antifungal drugs in clinical use. These agents inhibit β-glucan synthase, which catalyzes the synthesis of β-glucan, an essential component of the fungal cell wall, and have a high clinical efficacy and low toxicity. Echinocandin resistance is largely due to mutations in the gene encoding β-glucan synthase, but the mode of action is not fully understood. We developed fluorescent probes based on caspofungin, the first clinically approved echinocandin, and studied their cellular biology in Candida species, the most common cause of human fungal infections worldwide. Fluorescently labeled caspofungin probes, like the unlabeled drug, were most effective against metabolically active cells. The probes rapidly accumulated in Candida vacuoles, as shown by colocalization with vacuolar proteins and vacuole-specific stains. The uptake of fluorescent caspofungin is facilitated by endocytosis: The labeled drug formed vesicles similar to fluorescently labeled endocytic vesicles, the vacuolar accumulation of fluorescent caspofungin was energy-dependent, and inhibitors of endocytosis reduced its uptake. In a panel comprised of isogenic Candida strains carrying different β-glucan synthase mutations as well as clinical isolates, resistance correlated with increased fluorescent drug uptake into vacuoles. Fluorescent drug uptake also associated with elevated levels of chitin, a sugar polymer that increases cell-wall rigidity. Monitoring the intracellular uptake of fluorescent caspofungin provides a rapid and simple assay that can enable the prediction of echinocandin resistance, which is useful for research applications as well as for selecting the appropriate drugs for treatments of invasive fungal infections.We thank D. Perlin, D. Sanglard, R. Ben-Ami, C. Fairhead, B. Vincent, J. Usher, D. Soll, and S. Lindquist for providing the Candida strains. This work was supported by the Israel Science Foundation, Grant Nos. 179/19 (to M.F.) and 997/18 (to J.B.).Peer ReviewedPostprint (published version