bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.425432; this version posted March 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 High-throughput tandem-microwell assay for ammonia repositions 2 FDA-Approved drugs to Helicobacter pylori infection 3 Fan Liu,a,b,# Jing Yu,b,# Yan-Xia Zhang,c Fangzheng Li,a, d Qi Liu,e Yueyang Zhou,a 4 Shengshuo Huang,b Houqin Fang,f Zhuping Xiao,e Lujian Liao,f Jinyi Xu,d Xin-Yan Wu,c 5 Fang Wu a,* 6 7 aKey Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for 8 Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, 200240, China 9 bState Key Laboratory of Microbial Metabolism, Sheng Yushou Center of Cell Biology 10 and Immunology, School of Life Science and Biotechnology, Shanghai Jiao Tong 11 University, Shanghai, 200240, China 12 cSchool of Chemistry & Molecular Engineering, East China University of Science and 13 Technology, Shanghai, 200237, China. 14 dState Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, 15 China Pharmaceutical University, Nanjing, 210009, China 16 eHunan Engineering Laboratory for Analyse and Drugs Development of Ethnomedicine 17 in Wuling Mountains, Jishou University, Hunan, 416000, China 18 fShanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China 19 Normal University, Shanghai, 200241, China. 20 #These authors contributed equally to this work. 21 *To whom correspondence may be addressed. Email: [email protected] 22 23 Abbreviations: AHA, acetohydroxamic acid; CBS, cystathionine beta-synthase; CSE, cystathionine 24 -lyase; DTT, dithiothreitol; EBS, ebselen; FAD, Foreign Approved Drugs; FDA, U.S. Food and Drug 25 Administration; H. pylori, Helicobacter pylori; HPU, H. pylori urease; HTS, high-throughput 26 screening; JBU, jack bean urease; KD, equilibrium dissociation constant; LB, Luria-Bertani liquid 27 medium; MICs, minimum inhibitory concentrations; O. anthropi, Ochrobactrum anthropi; OAU, 28 Ochrobactrum anthropic urease; P. mirabilis, Proteus mirabilis; SPR, surface plasmon resonance. 29 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.425432; this version posted March 15, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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. 30 Abstract 31 To date, little attempt has been made to develop new treatments for Helicobacter 32 pylori (H. pylori), although the community is aware of the shortage of treatments for H. 33 pylori. In this study, we developed a 192-tandem-microwell-based high-throughput-assay 34 for ammonia that is a known virulence factor of H. pylori and a product of urease. We 35 could identify few drugs, i.e. panobinostat, dacinostat, ebselen, captan and disulfiram, to 36 potently inhibit the activity of ureases from bacterial or plant species. These inhibitors 37 suppress the activity of urease via substrate-competitive or covalent-allosteric mechanism, 38 but all except captan prevent the antibiotic-resistant H. pylori strain from infecting human 39 gastric cells, with a more pronounced effect than acetohydroxamic acid, a well-known 40 urease inhibitor and clinically used drug for the treatment of bacterial infection. This 41 study offers several bases for the development of new treatments for urease-containing 42 pathogens and to study the mechanism responsible for the regulation of urease activity. 43 44 45 46 47 Keywords: Ammonia, High-throughput screening, Antibiotic resistance, Urease, 48 Mechanism of action, Helicobacter pylori 49 50 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.425432; this version posted March 15, 2021. 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. 51 1. Introduction 52 Bacteria, fungi and plants, with the exception of animals, contain urease[1]. Urease (EC 53 3.5.1.5) is a class of nickel metalloenzyme that hydrolyzes amino acid metabolites to 54 produce ammonia (NH3) and carbon dioxide[2, 3]. The active catalytic site of urease 55 consists of two nickel ions, a carbamylated lysine residue, two histidines and an aspartic 56 acid. In addition to the consistent catalytic mechanism, the amino acid sequence of urease 57 has been reported to be highly conserved between different species[4]. 58 Bacterial urease is known to be a key virulence factor of some pathogens for a number of 59 diseases[5], e.g., Helicobacter pylori (H. pylori) for gastritis or gastric cancer, and 60 Proteus mirabilis (P. m i ra b i li s ) for urinary tract infections and urinary stones[6] . The 61 pathogens can hydrolyze urea substrates to produce NH3. The released NH3 not only 62 helps H. pylori to survive in the low pH environment of the stomach but also causes 63 damage to the gastric mucosa, triggering the infection[7]. Additionally, NH3 generated by 64 P. mirabilis urease has been demonstrated to form urinary stones and destroy the urinary 65 epithelium in the urinary system[8]. Because the human body does not contain urease, 66 bacterial urease has been thought to be an important and specific drug target for 67 combating these pathogens[9]. 68 A number of studies have been performed to identify inhibitors of urease[10-13], but only 69 one urease inhibitor, acetohydroxamic acid (AHA), was approved for the treatment of 70 urinary infections and urinary stones in 1983 by the US Food and Drug Administration 71 (FDA)[14, 15]. Severe side effects, low stability in gastric juice, and a lack of direct 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.425432; this version posted March 15, 2021. 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. 72 evidence for suppressing the growth of pathogens seem to be the limiting factors for the 73 low success rate of these urease inhibitors. Adverse side effects of AHA, including 74 teratogenic effects[16], a low efficiency indicated by the required high dose for the 75 patient (~ 1000 mg/day for adults), and the assumed drug resistance of bacteria, further 76 imply that potent and bioactive inhibitors with new chemical moieties are urgently 77 needed to combat these pathogens. Indeed, the current clinical first-line regimen for the 78 treatment of H. pylori [proton-pump inhibitor, clarithromycin, amoxicillin or 79 metronidazole (sometimes tinidazole)][17, 18], is unable to completely eradicate H. 80 pylori due to the increased antibiotic resistance[17, 19]. 81 To date, few validated high-throughput assay has been constructed to quantitatively 82 analyze NH3 and the activity of NH3-generating enzyme urease, but no high-throughput 83 screening approach has been employed to systematically extend the chemical moiety of 84 urease inhibitors. The current assay to determine the activity of urease mainly relies on 85 colorimetric reactions to determine the concentration of NH3 using indophenol or 86 Nessler’s reaction[20]. Recently, a microfluidic chip-based fluorometric assay has been 87 developed to monitor the activity of urease[21, 22]. In addition, a cell-based assay for H. 88 pylori urease has been reported lately, and validated by known inhibitors of urease, but it 89 has not been employed to screen new inhibitors for urease yet[23]. Overall, the current 90 assay setting and procedures are relatively time-consuming and vulnerable to 91 interference. 92 In this study, we established and validated a new tandem-well-based HTS assay for NH3 4 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.425432; this version posted March 15, 2021. 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. 93 and NH3-generating urease and performed an HTS screening campaign to identify 94 druggable chemical entities from 3,904 FDA or Foreign Approved Drugs (FAD) 95 -approved drugs for jack bean and bacterial ureases. Five clinically used drugs, i.e., 96 panobinostat, dacinostat, ebselen (EBS), captan and disulfiram, were found to be 97 submicromolar inhibitors of H. pylori urease (HPU), jack bean urease (JBU), or urease 98 from Ochrobactrum anthropi (O. anthropi), a newly identified pathogen with resistance 99 to -lactam antibiotics[24]. Moreover, panobinostat, dacinostat, EBS and disulfiram 100 potently inhibited the infection of H. pylori, suggesting that these pharmacologically 101 active moieties or drugs could serve as bases for the development of new treatments for 102 urease-positive pathogens. 5 bioRxiv preprint doi: https://doi.org/10.1101/2021.01.05.425432; this version posted March 15, 2021. 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. 103 2. Material and methods 104 2.1 Materials 105 Jack bean urease (JBU), DMSO, and dithiothreitol (DTT) were purchased from Sigma 106 (Steinheim, Germany). Hypochlorous acid, sodium nitroprusside, salicylate, potassium 107 sodium tartrate, urea, sodium hydroxide, bovine serum albumin, Triton X-100, 108 L-histidine and L-cysteine were purchased from Sangon (Shanghai, China).