20 21 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 1 9 / 2 0 Macromolecular Crystallography Group Beamline I24 A new target formalaria research Related publication: Baragaña B., Forte B., Choi R., Hewitt S. N., Bueren-Calabuig J. A., Pisco J. P., Peet C., DranowD. M., Robinson D. A., Jansen C., Norcross N. R.,Vinayak S., AndersonM., Brooks C. F., Cooper C. A., Damerow S., Delves M., Dowers K., Duffy J., EdwardsT. E., Hallyburton I., Horst B. G., HulversonM. A., Ferguson L., Jiménez-Díaz M. B., Jumani R. S., Lorimer D. D., LoveM. S., Maher S., Matthews H., McNamara C.W., Miller P., O’Neill S., Ojo K. K., Osuna-CabelloM., Pinto E., Post J., Riley J., RottmannM., Sanz L. M., Scullion P., Sharma A., Shepherd S. M., ShishikuraY., Simeons F. R. C., Stebbins E. E., Stojanovski L., Straschill U.,Tamaki F.K.,Tamjar J.,Torrie L. S.,Vantaux A.,Witkowski B.,Wittlin S.,Yogavel M., Zuccotto F., Angulo- Barturen I., Sinden R., Baum J., Gamo F-J., Mäser P., Kyle D. E.,Winzeler E. A., Myler P. J.,Wyatt P. G., Floyd D., Matthews D., Sharma A., Striepen B., Huston C. D., Gray D.W., Fairlamb A. H., Pisliakov A.V.,Walpole C., Read K. D.,VanVoorhisW. C. & Gilbert I. H. Lysyl-tRNA synthetase as a drug target inmalaria and cryptosporidiosis. P. Natl. Acad. Sci. USA 116 (14) 7015-7020 (2019). DOI:10.1073/pnas.1814685116 Publication keywords: Malaria; Cryptosporidiosis; tRNA synthetase;Target based drug discovery N ew drugs are needed to treat malaria, which caused more than 400,000 deaths worldwide in 2017. A related parasite causes the gastrointestinal disease cryptosporidiosis, which spreads through contact with human or animal faeces (often via dirty water). Although symptoms generally subside in a couple of weeks in healthy individuals, the disease can be fatal in patients with compromised immune systems. Estimates suggest that cryptosporidiosis causes more than 200,000 deaths each year, with malnourished children particularly at risk. At present, there is no effective treatment for infected children. Target-based screening is a focused approach to drug design, which looks for compounds that bind to, or inhibit, enzymes critical to disease. While it has been a successful approach for many other diseases, a lack of validated targets for themalaria parasite has hampered the use of target-based screening. A team of researchers at the University of Dundee, together with many collaborators, used Macromolecular Crystallography (MX) on the Microfocus and Serial MX beamline (I24), together with complementary techniques, to validate a novel biological target critical to both malaria and cryptosporidiosis and an exciting new compound series that shows activity against this target. Having identified a drug target for both malaria and cryptosporidiosis, and a series of compounds that inhibit it, the team’s focus now is on improving the properties of the compound series. It’s early days, but the goal is to deliver a drug candidate that can pass all the safety milestones required to advance to clinical development. Malaria and cryptosporidiosis are major burdens to both global health and economic development in many countries. Malaria caused more than 400,000 deaths in 2017, and cryptosporidiosis is estimated to cause more than 200,000 deaths a year. The spread of drug resistance is a growing concern for malaria treatment, and there is no effective treatment for malnourished or immunocompromised children infected with cryptosporidium. New treatments with novel mechanisms of action are needed for both diseases. However, at present there are few validated targets for drug discovery in malaria and even less for cryptosporidiosis. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum . Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl- tRNA synthetase ( Pf KRS1) 1 . Unfortunately, cladosporin is not amenable to development as a drug lead itself due to its high metabolic instability and lack of oral bioavailability. To identify new inhibitors of this promising target, as a collaboration with the University of Washington in Seattle, under the auspices of the Structure- guided Drug Discovery Coalition, recombinant Pf KRS1 was produced, assays developed and a biochemical screen of the GlaxoSmithKline malaria actives set of about 13,000 compounds was carried out (the Tres Cantos Antimalarial Set). The most promising hit for the screen showed similar levels of inhibition of Pf KRS1 and P. falciparum asexual blood stage as cladosporin. This hit also suffers from low metabolic stability like cladosporin but had the advantage of being synthetically tractable allowing rapid compound development. Thus, a hit optimisation project was started with the aim of developing analogues with improved metabolic stability and similar potency capable of clearing parasites frommouse models of malaria. The structural information gained using the beamline facilities at Diamond Light Source, to understand how the hit compound and analogues interact within the active site of the enzyme, has played a key role in the optimisation process. MX revealed the shape of molecules and insights into their function, and the crystallographers in the team worked at facilities in Seattle and New Delhi as well as at Diamond. The Dundee team have BAG (Block Allocation Group) access to Diamond, an access route specially designed for groups of users who require regular access, and who can coordinate different short experiments to fill a beamtime shift of eight hours. For this particular work, beamline I24 at Diamond was used. A published structure of cladosporin bound to Pf KRS1 showed that cladosporin binds within the ATP binding pocket 2 . The screening hit was co- crystallised with Pf KRS1 and also binds in the ATP binding pocket. The core of the hit occupies the same space as the adenine ring of ATP and the cyclohexyl moiety projects into the pocket where the ribose ring of ATP binds. This pocket is completed by the substrate lysine. Several rounds of compound design, aided by the structural information and computational models, followed by synthesis and testing led to the identification of a lead molecule with similar potency and selectivity and excellent metabolic stability. The complex of the lead bound to the enzyme showed that the changes introduced to improve metabolic stability had minimal effect upon the position of the ligand within the binding site with respect to hit compound (Fig. 1). The lead compound was active against both Pf KRS1 (IC 50 = 0.015 μM) and whole-cell bloodstream P. falciparum 3D7 (EC 50 = 0.27 μM) and was selective compared with both the Hs KRS (IC 50 = 1.8 μM) and HepG2 cells (EC 50 = 49 μM). The biological and pharmacokinetic profile was sufficient to justify a rodent efficacy study. The lead was evaluated in vivo in a mouse model of malaria, which showed a reduction of the number of malaria parasites in blood by 90% at day 5 of the study. There is a high level of sequence identity within the active-site regions of Pf KRS1 and Cp KRS. Therefore, cladosporin, the screening hit, and the lead compound were tested in a cellular assay against C. parvum . The three compounds showed inhibition of parasite growth. Several structures of C. parvum enzyme bound to our inhibitors showed retention of the ligand binding mode compared with the malaria enzyme.These results prompted the researchers to progress the lead compound to in vivo efficacy in two different Cryptosporidium mouse models. A reduction of parasite burden by two orders of magnitude with the lead compound in both disease models was observed. Furthermore, X-ray crystallography and molecular dynamics simulations were used to rationalise the selectivity of the compounds for Pf KRS1 and Cp KRS compared to (human) Hs KRS. MD simulations suggest that the selectivity observed for the lead is due to a combination of a more favourable configuration of the binding site and a higher degree of stabilisation upon ligand binding in the parasite enzymes. These results offer a strong validation of lysyl-tRNA synthetase as a drug target for malaria and cryptosporidiosis. References: 1. Hoepfner D. et al . Selective and specific inhibition of the plasmodium falciparum lysyl-tRNA synthetase by the fungal secondary metabolite cladosporin. Cell Host Microbe 11 (6), 654-663 (2012). DOI: 10.1016/j.chom.2012.04.015 2. Khan S. et al . Structural basis of malaria parasite lysyl-tRNA synthetase inhibition by cladosporin. J. Struct. Funct. Genomics. 15 (2), 63-71 (2014). DOI: 10.1007/s10969-014-9182-1 Funding acknowledgement: We thank the European Synchrotron Radiation Facility for beamtime, highlighting the staff of beamlines BM14 and ID29; Diamond Light Source for beamtime (proposal mx10071); the staff of beamline I24 for assistance with crystal testing and data collection; the entire Seattle Structural Genomics Center for Infectious Disease team; the Division of Biological Chemistry and Drug Discovery Protein Production Team; GlaxoSmithKline for the Tres Campos Antimalarial screening set; the Scottish Blood Transfusion Centre (Ninewells Hospital, Dundee) for providing human erythrocytes; Christoph Fischli and Sibylle Sax at the SwissTPH for technical assistance with the SCID mouse model; and Anja Schäfer for technical assistance with the in vitro antimalarial activity testing. The Art of Discovery thanks Dr. Cristina Eguizabal and the Basque Center of Transfusion and Human Tissues (Galdakao, Spain) and the Bank of Blood and Tissues (Barcelona, Spain) for providing human blood. The University of California, San Diego thanks Jenya Antonova-Koch for help. This work was supported by the Bill and Melinda Gates Foundation through Grant OPP1032548 to the Structure-Guided Drug Discovery Coalition and OPP1134302 (to B.S.). This work was also supported in part from federal funds, from the NIH/National Institute of Allergy and Infectious Diseases Grant R21AI123690 (to K.K.O.) and Contracts HHSN272201200025C and HHSN272201700059C (to P.J.M.); Medicines for Malaria Venture (through access to assays to I.H.G. and through RD/08/2800 to J.B.); Wellcome Trust for support of the X-ray Crystallography Facility 094090, IT support Grant 105021 (to I.H.G.), and Institutional Strategic Support Fund 204816 (to A.V.P.), all at the University of Dundee and for Investigator Award 100993 (to J.B.). Corresponding authors: Prof Ian H. Gilbert, University of Dundee,
[email protected] ; Dr Beatriz Baragana, University of Dundee,
[email protected] Figure 1: A co-crystal structure of the lead compound with P. falciparum lysysl-tRNA synthetase.