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The Transcriptome of Balamuthia Mandrillaris Trophozoites for Structure-Based Drug 2 Design 3 Isabelle Q

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The Transcriptome of Balamuthia Mandrillaris Trophozoites for Structure-Based Drug 2 Design 3 Isabelle Q bioRxiv preprint doi: https://doi.org/10.1101/2020.06.29.178905; this version posted June 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 The transcriptome of Balamuthia mandrillaris trophozoites for structure-based drug 2 design 3 Isabelle Q. Phan1,2*Δ, Christopher A. Rice3,†*Δ, Justin Craig1,4, Rooksana E. Noorai5, Jacquelyn 4 McDonald2, Sandhya Subramanian1,2, Logan Tillery1,4, Lynn K. Barrett1,4, Vijay Shankar6, James 5 C. Morris7, Wesley C. Van Voorhis1,4,8,9 Dennis E. Kyle3, Peter J. Myler1,2,9,10Δ 6 1Seattle Structural Genomics Center for Infectious Disease (SSGCID), Seattle, Washington, USA. 7 2Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, 8 Washington, USA. 9 3Center for Tropical and Emerging Global Diseases, University of Georgia, Athens, Georgia, USA. 10 4Center for Emerging and Re-emerging Infectious Diseases (CERID), Division of Allergy and 11 Infectious Diseases, Department of Medicine, University of Washington, Seattle, Washington, 12 USA. 13 5Clemson University Genomics and Bioinformatics Facility, Clemson University, Clemson, South 14 Carolina, USA. 15 6Center for Human Genetics, Clemson University, Greenwood, South Carolina, USA. 16 7Eukaryotic Pathogens Innovation Center, Department of Genetics and Biochemistry, Clemson 17 University, Clemson, South Carolina, USA. 18 8Department of Microbiology, University of Washington, Seattle, Washington, USA. 19 9Department of Global Health, University of Washington, Seattle, Washington, USA. 20 10Department of Pediatrics, University of Washington, Seattle, Washington, USA. 21 †Current Address: Department of Pharmaceutical and Biomedical Sciences, College of 22 Pharmacy, University of Georgia, Athens, Georgia, USA. 23 * Authors contributed equally to this work. 24 25 Δ Author correspondence: [email protected] (I.Q.P), [email protected] 26 (C.A.R) and [email protected] (P.J.M). 27 28 29 30 31 32 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.29.178905; this version posted June 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 33 Abstract: 34 Balamuthia mandrillaris, a pathogenic free-living amoeba (FLA), causes cutaneous skin lesions 35 as well as the brain-eating disease: Balamuthia granulomatous amoebic encephalitis (GAE). 36 These diseases, and diseases caused by other pathogenic FLA, Naegleria fowleri or 37 Acanthamoeba species, are minimally studied from a drug discovery perspective; few targets 38 have been validated or characterized at the molecular level, and little is known about the 39 biochemical pathways necessary for parasite survival. Chemotherapies for CNS disease caused 40 by B. mandrillaris require vast improvement. Current therapeutics are limited to a small number 41 of drugs that were previously discovered in the last century through in vitro testing or identified 42 after use in the small pool of surviving reports. 43 Using our recently published methodology to identify potentially useful therapeutics, we 44 screened a collection of 85 compounds that have previously been reported to have antiparasitic 45 activity. We identified 59 compounds that impacted growth at concentrations below 220 µM. 46 Since there is no fully annotated genome or proteome, we used RNA-Seq to reconstruct the 47 transcriptome of B. mandrillaris and locate the coding sequences of the specific genes potentially 48 targeted by the compounds identified to inhibit trophozoite growth. We determined the sequence 49 of 17 of these target genes and obtained expression clones for 15 that we validated by direct 50 sequencing. 51 Introduction 52 Balamuthia mandrillaris is a ubiquitous soil-dwelling amoeba that is the causative agent of 53 GAE1–4. In common with the other two major pathogenic free-living amoebas (FLAs), Naegleria 54 fowleri and Acanthamoeba castellanii, B. mandrillaris infections, though uncommon, have > 55 90% case fatality rate5. In the United States, 109 Balamuthia cases in both immunocompetent 56 and immunocompromised individuals have been reported with at least twice that many 57 worldwide, but these rates of GAE are likely to be underestimated due to historically poor 58 diagnosis6. Nonetheless, awareness of potential cases has been on the rise7,8. In addition to 59 diagnostic awareness, increasing rates of amoebic infections in northern regions of the United 60 States could also be early indicators that recent emergence of these diseases might be associated 61 with global warming6. In contrast to Naegleria infections that present and progress extremely 62 rapidly after exposure, Balamuthia incubation times might be as long as several months and 63 disease progression more subacute or chronic, increasing the opportunity for therapeutic 64 intervention9. However, treatment options remain very limited, leading to poor outcomes even 65 with the correct diagnosis. 66 67 The recent development of an inexpensive and easily prepared media, as well as increasing 68 interest in B. mandrillaris as a public health concern, has facilitated the development of robust 69 high-throughput drug screening methods. Where low throughput methods restricted screening to 70 ~10-20 drugs at a time, the new high-throughput methods allow rapid screening of hundreds to 71 thousands of drugs simultaneously and allows direct comparisons of activity. In this study, we 72 identify FDA approved drugs that could potentially be repurposed for therapy alone or added bioRxiv preprint doi: https://doi.org/10.1101/2020.06.29.178905; this version posted June 30, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 73 into a combination drug cocktail against B. mandrillaris. These drugs can also be used as leads 74 for further structure-based drug discovery (SBDD) exploration and in vivo efficacy studies. 75 Structure based drug discovery (SBDD) was originally devised in the mid 1980’s10. With 76 advances in methodology for protein structure elucidation, less expensive and faster computer 77 processing, and improved access to prediction software, the timeline of solving target structures 78 and developing specific and selective drugs have significantly shortened10. SBDD has been 79 discussed by several authors throughout the amoeba literature as an attractive method of 80 designing selective enzymatic inhibitors that would specifically target the parasite over the 81 human host11. Given how frequently this strategy is discussed, it is surprising that only a small 82 number of laboratories have actually tested this methodology in practice against pathogenic 83 FLA. Sterol biosynthesis has been the most attractive target since parasites utilize ergosterol over 84 cholesterol for making cell plasma membranes with distinct host biosynthetic differences that 85 could be selectively targeted12–14. Glucose metabolism is essential for parasitic cell viability. 86 Milanes et al., recently targeted glucokinase in Naegleria fowleri, and described NfGlck specific 87 inhibitors with minimal activity against human glucokinase in recombinant enzymatic functional 88 studies15. Other studies have looked at targeting histidine or shikimate essential amino acid 89 biosynthetic pathways in Acanthamoeba species, which the hosts cannot synthesize de novo, as 90 parasite specific targets for drug intervention16,17. 91 The development of new compounds against B. mandrillaris in particular has been hampered by 92 the paucity of genomic information. Though draft genomes have been published, no structural 93 and functional annotation is currently available18,19. This information is essential for the design 94 of new drugs by SBDD, as that methodology requires information about the molecular structure 95 of the target protein. Once the protein coding sequences are annotated on the genome, rapid 96 selection of multiple drug targets can be performed, for example by homology searches with 97 known drug targets, thus paving the way for combinational therapy, a broadly established 98 strategy to minimize the risk of drug resistance. This study presents the first comprehensive 99 proteome of B. mandrillaris reconstructed from RNA sequencing of logarithmic growing 100 trophozoites, the infective form of the amoeba. Potential drug targets identified through 101 phenotypic screening were selected specifically from the trophozoite transcriptome and PCR 102 amplified. The clones were further validated by direct sequencing, providing the first step for 103 recombinant expression and crystallization by the Seattle Structural Genomics Center for 104 Infectious Disease (SSGCID) high-throughput gene-to-structure pipeline20. 105 Results and discussion 106 Phenotypic screens 107 We performed a drug susceptibility screen of 85 compounds, previously identified as 108 antiparasitic, against the trophozoite stage of B. mandrillaris, and discovered that 59 of these 109 compounds had 50% inhibitory concentration (IC50) efficacy at ≤ 220 µM concentration (Table 110
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