Microsoft Word - Urease_inhibitor_actanew2 1    High-throughput tandem-microwell assay for ammonia repositions 1  FDA-Approved drugs to Helicobacter pylori infection 2  Fan Liu,a,b,# Jing Yu,b,# Yan-Xia Zhang,c Fangzheng Li,a, d Qi Liu,e Yueyang Zhou,a 3  Shengshuo Huang,b Houqin Fang,f Zhuping Xiao,e Lujian Liao,f Jinyi Xu,d Xin-Yan Wu,c 4  Fang Wu a,* 5  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: fang.wu@sjtu.edu.cn 22  23  Running title: Repositioning of old drugs to treat H. pylori infection 24  25  .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 2    ABSTRACT 26  To date, little attempt has been made to develop new treatments for Helicobacter 27  pylori (H. pylori), although the community is aware of the shortage of treatments for H. 28  pylori. In this study, we developed a 192-tandem-microwell-based high-throughput-assay 29  for ammonia that is a known virulence factor of H. pylori and a product of urease. We 30  could identify few drugs, i.e. panobinostat, dacinostat, ebselen, captan and disulfiram, to 31  potently inhibit the activity of ureases from bacterial or plant species. These inhibitors 32  suppress the activity of urease via substrate-competitive or covalent-allosteric mechanism, 33  but all except captan prevent the antibiotic-resistant H. pylori strain from infecting human 34  gastric cells, with a more pronounced effect than acetohydroxamic acid, a well-known 35  urease inhibitor and clinically used drug for the treatment of bacterial infection. This 36  study offers several bases for the development of new treatments for urease-containing 37  pathogens and to study the mechanism responsible for the regulation of urease activity. 38  39  Key Words: Ammonia, High-throughput screening, Antibiotic resistance, Enzyme 40  inhibitor, Urease, Mechanism of action, Helicobacter pylori 41  42  43  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 3    INTRODUCTION 44  Bacteria, fungi and plants, with the exception of animals, contain urease(1). Urease (EC 45  3.5.1.5) is a class of nickel metalloenzyme that hydrolyzes amino acid metabolites to 46  produce ammonia (NH3) and carbon dioxide(2,3). The active catalytic site of urease 47  consists of two nickel ions, a carbamylated lysine residue, two histidines and an aspartic 48  acid. In addition to the consistent catalytic mechanism, the amino acid sequence of urease 49  has been reported to be highly conserved between different species(4). 50  Bacterial urease is known to be a key virulence factor of some pathogens for a number of 51  diseases(5), e.g., Helicobacter pylori (H. pylori) for gastritis or gastric cancer, and 52  Proteus mirabilis (P. mirabilis) for urinary tract infections and urinary stones(6) . The 53  pathogens can hydrolyze urea substrates to produce NH3. The released NH3 not only 54  helps H. pylori to survive in the low pH environment of the stomach but also causes 55  damage to the gastric mucosa, triggering the infection(7). Additionally, NH3 generated by 56  P. mirabilis urease has been demonstrated to form urinary stones and destroy the urinary 57  epithelium in the urinary system(8). Because the human body does not contain urease, 58  bacterial urease has been thought to be an important and specific drug target for 59  combating these pathogens(9). 60  A number of studies have been performed to identify inhibitors of urease(10-12), but only 61  one urease inhibitor, acetohydroxamic acid (AHA), was approved for the treatment of 62  urinary infections and urinary stones in 1983 by the US Food and Drug Administration 63  (FDA)(13,14). Severe side effects, low stability in gastric juice, and a lack of direct 64  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 4    evidence for suppressing the growth of pathogens seem to be the limiting factors for the 65  low success rate of these urease inhibitors. Adverse side effects of AHA, including 66  teratogenic effects(15), a low efficiency indicated by the required high dose for the 67  patient (~ 1000 mg/day for adults), and the assumed drug resistance of bacteria, further 68  imply that potent and bioactive inhibitors with new chemical moieties are urgently 69  needed to combat these pathogens. Indeed, the current clinical first-line regimen for the 70  treatment of H. pylori [proton-pump inhibitor, clarithromycin, amoxicillin or 71  metronidazole (sometimes tinidazole)](16,17), is unable to completely eradicate H. pylori 72  due to the increased antibiotic resistance(16,18). 73  To date, few validated high-throughput assay has been constructed to quantitatively 74  analyze NH3 and the activity of NH3-generating enzyme urease, but no high-throughput 75  screening approach has been employed to systematically extend the chemical moiety of 76  urease inhibitors. The current assay to determine the activity of urease mainly relies on 77  colorimetric reactions to determine the concentration of NH3 using indophenol or 78  Nessler’s reaction(19). Recently, a microfluidic chip-based fluorometric assay has been 79  developed to monitor the activity of urease(20,21). In addition, a cell-based assay for H. 80  pylori urease has been reported lately, and validated by known inhibitors of urease, but it 81  has not been employed to screen new inhibitors for urease yet(22). Overall, the current 82  assay setting and procedures are relatively time-consuming and vulnerable to 83  interference. 84  In this study, we established and validated a new tandem-well-based HTS assay for NH3 85  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 5    and NH3-generating urease and performed an HTS screening campaign to identify 86  druggable chemical entities from 3,904 FDA or Foreign Approved Drugs (FAD) 87  -approved drugs for jack bean and bacterial ureases. Five clinically used drugs, i.e., 88  panobinostat, dacinostat, ebselen (EBS), captan and disulfiram, were found to be 89  submicromolar inhibitors of H. pylori urease (HPU), jack bean urease (JBU), or urease 90  from Ochrobactrum anthropi (O. anthropi), a newly identified pathogen with resistance 91  to -lactam antibiotics(23). Moreover, panobinostat, dacinostat, EBS and disulfiram 92  potently inhibited the infection of H. pylori, suggesting that these pharmacologically 93  active moieties or drugs could serve as bases for the development of new treatments for 94  urease-positive pathogens.95  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 6    RESULTS 96  Development of a high-throughput assay and identification of potent inhibitors for 97  urease 98  To construct a high-throughput assay for NH3-generating urease and prevent the detection 99  interference from substances in the enzyme extraction, we utilized a 100  192-tandem-well-based gas-detection method, which we previously developed to monitor 101  the activity of H2S-generating enzymes(24,25). The tandem-well design could physically 102  separate the gas product from the enzymatic reaction and enable the specific and 103  real-time detection of the gas-producing enzyme activity (Figure 1A). 104  To construct the HTS assay, we compared three reported protocols for determination of 105  the activity of JBU by using salicylic acid-hypochlorite and Nessler detection reagent, as 106  well as phenol red(20,26,27), which undergo the indophenol and Nessler’s reaction with 107  NH3, respectively. Salicylic acid-hypochlorite and Nessler’s reagents could 108  dose-dependently and time-dependently monitor the activity of JBU at various 109  concentrations (Figures S1A and B); however, the phenol red failed to detect it (Figure 110  S1C). We decided to choose salicylic acid-hypochlorite as the detection reagent for the 111  HTS screening assay of JBU (Figure S1A) due to its lower toxicity than Nessler reagent, 112  which contains mercury(26). The absorbance (OD) at 697 nm of the blue complex 113  indophenol generated from salicylic acid was correlated linearly with the concentration of 114  NH4Cl (19.5 - 625 M), thus validating the analytic setup for NH3 quantification (Figure 115  S1D). Moreover, the optimal assay buffer for JBU was found to be phosphate buffer at 116  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 7    pH 7.4 (Figure S1E). In contrast, we employed Nessler’s reagent to detect the activity of 117  HPU and Ochrobactrum anthropic urease (OAU) in subsequent studies since it showed a 118  better sensitivity for the limitation of detection of the activity of HPU than salicylic 119  acid-hypochlorite (Figures S1F and 1G). Collectively, we chose 50 nM of JBU and 25 120  mM urea substrate in the phosphate buffer to perform the assay. 121  Under the assay conditions, AHA showed an IC50 of ~ 160 μM (Figure 1B), which was 122  very similar to the previously reported value (IC50 of ~ 140 μM; ref. (13)), indicating that 123  the newly developed assay for urease was accurate and reliable. However, the IC50 of 124  AHA was found to decrease to 33.7 μM when using the 50 mM Tris buffer instead of the 125  phosphate buffer in our assay (Table 1). To determine the well-to-well reproducibility, the 126  assay was validated with 200 M AHA (~ IC50) or 800 M (~ 5-fold IC50) AHA. The 127  tandem-well plate consistently showed distinct differences among the control, the 200 128  M-AHA-treated and the 800 M-AHA-treated groups (Figure 1C). The average Z’ 129  values of the assay were found to be ~ 0.9 when they were calculated with the 800 M 130  AHA positive control. 131  To identify novel and potent inhibitors for urease, we screened 3,904 FDA or FAD 132  -approved drugs at 100 μM. Five potent hits, i.e., panobinostat, dacinostat, EBS, captan 133  and disulfiram, were found to dose-dependently inhibit the activity of JBU with IC50 134  values of 0.2, 1.1, 0.4, 2.3 and 38.9 M, which are ~ 800, 146, 400, 70, 4, -fold more 135  potent than AHA, respectively (Figure 1E and Table 1). Intriguingly, the former two 136  drugs are analogs of AHA. Importantly, all of them seemed to bear significant 137  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 8    selectivities for urease since they did not substantially inhibit other gas-producing 138  enzymes, i.e., cystathionine beta-synthase (CBS) and cystathionine -lyase (CSE), two 139  H2S-generating enzymes (Figure 1F). Moreover, the potent inhibitory effects of these 140  inhibitors were likely due to on-target inhibition of JBU rather than the nonspecific 141  reaction with NH3 or forming an aggregation since they did not react with NH3 and their 142  inhibition was not attenuated by the detergent (Figures S2A and S2B). In corroborating 143  these findings, EBS and disulfiram have recently been reported to be specific inhibitors 144  of bacterial and plant urease(11,12), respectively, although their mode of actions for 145  inhibiting urease, and their effects on the proliferation or infection of urease-containing 146  pathogens remain little explored. 147  148  The mode of action study for urease inhibitors 149  To determine the reversibility of the inhibition by panobinostat, dacinostat, EBS, captan 150  and disulfiram to JBU, various concentrations of the inhibitors and JBU were incubated 151  together for 60 min (Figure 2A). After a 200-fold dilution, the inhibitory effects of 152  panobinostat and dacinostat as well as disulfiram were found to be reversible (Figures 2A 153  and S3C). In contrast, EBS or captan at 100 nM was found to completely block the 154  activity of JBU; this concentration did not affect the activity without the pre-incubation 155  with enzyme (Figure 1E). Additionally, the inhibitions exerted by EBS or captan were not 156  fully recovered (Figure 2A), indicating that both of them were likely to be covalent or 157  slow-dissociation inhibitors for JBU. 158  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 9    Surprisingly, the inhibitory effect of disulfiram was found to be dependent on the 159  concentrations of Ni2+ ion, the catalytic cofactor for urease (Figure S3C), indicating that 160  it inhibits JBU likely via formation of a complex with the catalytic Ni2+ ion and 161  subsequently occupying the active site of JBU. This explanation seems to be plausible 162  since recent findings have revealed that disulfiram inhibits the proliferation of tumor cells 163  by forming a complex with Cu2+(28). 164  Moreover, the inhibitory potencies of panobinostat and dacinostat were found to increase 165  with the pre-incubation time of the compound with urease (Figure 2B). After 2 h 166  pre-incubation, the IC50 value of panobinostat and dacinostat were decreased ~ 7.5 folds 167  and ~ 18.8 folds, respectively (Figure 2B). In enzyme kinetics studies for JBU, 168  panobinostat and dacinostat were found to be competitive inhibitors towards urea 169  substrate, with a Ki value of 0.02 and 0.07 M (Figure 2C and Table 1), which are ~ 105 170  folds and 30 folds more potent than AHA (Ki ~ 2.1 M; Table 1). In consistent with this 171  observation, the inhibition of these two inhibitors doesn’t be interfered with Ni2+ (Figure 172  S3A). Also, the addition of histidine or cysteine has no effects on the inhibition of 173  panobinostat or dacinostat (Figure S3B). Importantly, the surface plasmon resonance 174  assay demonstrate that these two compounds could physically bind to JBU (Figure 2D; 175  Table 1). The drastic effect seems not only relying on the hydroxamic acid motif that is 176  the known pharmacophore of AHA-derivative inhibitors, but also the hydrophobic ring 177  and secondary amine group, as indicated by that the benzene ring favorably interacts with 178  the His492 residue and/or the nitrogen atom forms an additional hydrogen bond with 179  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 10    Asp494 in the modeled inhibitor-JBU complex structure (Figure 2E). 180  In contrast, the inhibition caused by both EBS and captan was found to be prevented by 181  the addition of dithiothreitol (DTT) or free cysteine into the enzymatic reaction, but not 182  that of histidine or Ni2+ (Figures S4A-C). Furthermore, the IC50 values of the two 183  inhibitors were linear with the concentrations of the enzyme (Figure S4D), an inhibitory 184  feature of the covalent inhibitor(29), confirming that they targeted the enzyme covalently. 185  The inhibition constants for these irreversible inhibitors, i.e., the rate of enzyme 186  inactivation (kinact) and inactivation rate constants (KI), were also determined by 187  nonlinear regression of the time-dependent IC50 values (Figure S4E)(29). The kinact and KI 188  for EBS were found to be 2.79 × 10-3 s-1 and 0.73 M, which were 4.4 and 2.4-fold better 189  than captan (kinact, 0.63 × 10 -3 s-1; KI, 1.76 M), respectively. Taken together, the results 190  demonstrated that EBS and captan inhibited JBU by covalently modifying the Cys rather 191  than His residue, the latter of which is known to be the active site of urease (2,3). 192  Interestingly, we observed a synergistic inhibitory effect from the combination of EBS 193  and AHA (Figure 3A), a substrate-competitive inhibitor for urease, implying that EBS 194  targeted Cys residue(s) of another site rather than the active site. Similar experimental 195  results were also obtained for captan. Moreover, the combination of EBS with 2 M 196  captan also significantly increased the potency of EBS by 6-fold (right panel, Figure 3A), 197  implying distinct binding sites of the two covalent inhibitors. 198  To corroborate this finding, we performed enzyme kinetics, mass spectrometry and 199  surface plasmon resonance studies (Figures 3B-D). Consistently, EBS or captan displayed 200  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 11    a noncompetitive mode for the urea substrate (Figure 3B). Furthermore, tandem-mass 201  spectrometry analysis revealed that Cys313 and Cys406, which were not adjacent to the 202  active site, appeared to be modified by EBS and captan, respectively (Figure 3C). The 203  addition of 274.18 daltons in molecular weight was observed for EBS, demonstrating the 204  breakage of the Se-N bond and formation of the Se-S bond with the Cys residue, a 205  phenomenon that has been reported previously for EBS(30). However, the increase of 206  150.15 daltons suggested that only the isoindole dione moiety of captan modified the Cys 207  residue, accompanied by the release of the trichloromethyl thio moiety [-SC(C1)3]. This 208  new observation provides a new perspective for the unexplored covalent molecular 209  mechanism of captan. 210  Additionally, a potent and physical interaction between EBS or captan and JBU was 211  observed in the surface plasmon resonance study (Figure 3D). The equilibrium 212  dissociation constant (KD) for EBS and captan was found to be 89 and 96 nM, 213  respectively. 214  To illustrate the binding mode of EBS or captan, we modeled them into the respective 215  allosteric Cys-containing pocket (Cys313 for EBS, Cys406 for Captan) in JBU by using 216  molecular dynamics simulations (Figure 3E). The carbonyl group of EBS was found to 217  form a hydrogen bond with Lys369, and the phenyl ring interacts with the hydrophobic 218  side chain of Leu308. Additionally, the two carbonyl groups of captan formed four 219  hydrogen bonds with the side chains of Asn517, His542, Tyr544 and Asn688. Taken 220  together, these results implied that these intermolecular weak interactions also 221  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 12    substantially contributed to the binding of the covalent inhibitors to the protein, in 222  addition to the covalent interaction. 223  The inhibitory effect of inhibitors on bacterial ureases 224  Next, we investigated the effects of panobinostat, dacinostat, EBS and captan as well as 225  disulfiram on the activity of HPU and OAU, two bacterial ureases from H. pylori and O. 226  anthropic, respectively. As expected, these drugs could inhibit the activity of HPU in the 227  crude extracts and showed IC50 values of 0.1 M, 0.2 M, 2.8 M, 3.4 and 8.9 M, 228  which indicated that they were ~ 259, 130, 10, 8 and 3 -fold more potent than AHA (IC50 229  ~ 25.9 M; Figure 4A and Table 1), respectively. Moreover, panobinostat, dacinostat, 230  EBS, captan and disulfiram were also found to inhibit the partially purified HPU, which 231  was isolated by size-exclusion chromatography (Figures 4B and S5). Consistently, they 232  also suppressed the activity of OAU at a similar potency to HPU (Figure 4A and Table 1). 233  Compounds 1, 4 and 6, which were synthesized in house (Scheme S1), as well as 234  commercially available EBS oxide, also showed a better efficiency than EBS (IC50 ~ 2.8 235  M) in the in vitro HPU-based enzyme assay (Table S1), and 4 displayed a maximum 236  three-fold increase in potency (IC50 ~ 1.1 M; Table S1). Moreover, we could confirm 237  that panobinostat, dacinostat and EBS as well as EBS oxide, 1, 4 or 6, could largely 238  suppress the activity of HPU in culture (Figure 4C). The IC50 values of these inhibitors 239  for inhibiting the urease of the cultured H. pylori strain ranged from 5.7 to 23.2 M 240  (Figure 4C and Table S2). 241  Further, we investigated the effects of panobinostat, dacinostat and EBS, which are the 242  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 13    most potent inhibitors for HPU (Figure 4A). The results showed that EBS, but not 243  panobinostat, dacinostat or its analog AHA, has a substantial suppression on the growth 244  of H. pylori (Figure 4D). The inability of AHA as well as its derivatives, i.e. panobinostat 245  and dacinostat, on the growth of H. pylori as identified above seems to be consistent with 246  the previous finding that AHA doesn’t inhibit the growth of H. pylori(31). Interestingly, 247  EBS and EBS analogs, as well as disulfiram, could dose-dependently suppress the growth 248  of H. pylori and showed a minimum inhibitory concentration (MIC) in a range between 2 249  and 4 g/ml (right panel of Figure 4D, Figure S6A and Table S2). Importantly, the 250  inhibitory effect of this type of covalent inhibitors lasted for a long period in culture, as 251  indicated by EBS and 1, which could substantially inhibit HPU even after removal of the 252  inhibitor for 6 h (Figure S6B). 253  Urease inhibitors prevent H. pylori infection in a gastric cell-based bacterial 254  infection model 255  To evaluate the ability of these urease inhibitors to prevent H. pylori infection, we 256  constructed a gastric cell-based bacterial infection model using the remaining viable cell 257  number of SGC-7901 adenocarcinoma gastric cells to reflect the virulence of H. 258  pylori(15). Our results showed that treatment with 30 M panobinostat, 30 M dacinostat, 259  20 M EBS or 20 M disulfiram could prevent the cell death triggered by H. pylori 260  (Figures 5A-B). In sharp contrast, the cells that lacked such treatments were largely 261  sabotaged. Panobinostat and EBS were found to be the most potent agents and almost 262  completely protected from the infection of H. pylori. These effects of these drugs seemed 263  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 14    to be much more efficient than the effects of 20 M AHA or 50 M tinidazole, the analog 264  of metronidazole, and one of the two antibiotics in the triple regimens for the treatment of 265  H. pylori (16,17). In support of this observation, tinidazole as well as metronidazole 266  hardly suppressed the growth of our H. pylori strain, with an MIC value of more than 512 267  g/ml in culture (Figure S7A and Table S2), indicating that this strain is resistant to 268  treatment with nitroimidazole-type antibiotics. 269  Since panobinostat, dacinostat, EBS and disulfiram at a concentration up to 100 M or 25 270  M did not interfere with the proliferation of SGC-7901 gastric cells (Figure S7B), the 271  protective effects in the gastric-cell-based H. pylori infection model seemed to be 272  attributed to on-targeting inhibition of the infection transmitted by H. pylori. Moreover, 273  all four drugs potentially inhibited the level of ammonia in the cell medium (Figure 5C), 274  indicating that they efficiently suppressed the endogenous urease activity of H. pylori in 275  the infection model. 276  277  The structural basis and inhibitory mechanisms of newly-identified three classes 278  urease inhibitors 279  To identify the active chemical moiety of panobinostat, dacinostat, EBS or captan 280  required for inhibition of urease, we analyzed their structure-activity relationships 281  (Figures 6, Table S1 and S3). The former two inhibitors are hydroxamic acid-based 282  urease inhibitors, and not only their hydroxyamino heads are forming hydrogen bonds 283  with the catalytic Ni2+ and residues in JBU or HPU (Asp633 or Ala636 for JBU; Asp362 284  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 15    or Ala365 for HPU), but also the acetyl group constitutes one hydrogen bond (His492 for 285  JBU and His221 for HPU; Figures 2E and S8A). Consistent with this observation, the 286  hydroxyamino and acetyl groups of AHA interact with Asp362 or Ala365 and His221 in a 287  co-crystal structure of AHA and HPU(2), respectively (Figure S8A). Compound lacking 288  of this acetyl group, i.e. hydroxylamine, totally abolished the inhibitory effect of this type 289  inhibitor (Figure 6B and Table S3). Apart from these interactions, the hydrophobic 290  benzene ring and secondary amine group of panobinostat were found to be additional 291  pharmacophores (upper panel, Figure 2E), which interact favorably with His492 (JBU) or 292  His221 (HPU) and form an extra hydrogen bond with Asp494 (JBU) or Asp223 (HPU). 293  In supporting this finding, the hydroxamic acid analogs that are lack of the benzene ring, 294  i.e. ricolinostat, ilomastat and pracinostat, are inactive to JBU and HPU (Figure 6B and 295  Table S3). Strikingly, the replacement of benzene with benzimidazole (pracinostat) totally 296  loses the inhibition, suggesting the benzene is critical for maintaining the inhibition. 297  Moreover, the secondary amine group seems to be also important for enhancing the 298  potency of this type inhibitor, since the modification or replacement of it with hydroxyl 299  group or sulfonyl group (dacinostat or belinostat), also weaken ~ 5-fold or 24-fold in IC50 300  values. 301  For EBS analogs, compounds (2-3) lacking the Se atom largely lost inhibitory activities 302  toward JBU and HPU (Figure 6B and Table S1). Furthermore, dibenzyl diselenide was 303  also inactive toward both ureases, indicating that the Se-containing benzisoxazole moiety 304  rather than the solo Se atom might be essential for the inhibition. Indeed, Se-containing 305  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 16    benzisoxazole (4) showed potent inhibition of HPU (IC50 ~ 0.8 and 1.1 M for JBU and 306  HPU, respectively). The introduction of an electron-donating group to the benzisoxazole 307  moiety apparently strongly reduced the potency (5; IC50 ~ 1.4 M for JBU and more than 308  10 M for HPU; Figure 6B). In contrast, the provision of electron-withdrawing groups to 309  the nitrogen or Se atom of the benzisoxazole moiety, i.e., 6 or EBS oxide, seemed to 310  enhance the potency of JBU by a maximum of three-fold (6). Similarly, when weakening 311  the electron-withdrawing effect in the substitution group of the isoindole dione core of 312  captan, the active moiety (Figure 3C), was also found to lead to a decreased potency 313  (Figures 6; Table S1). Taken together, these data indicate that the Se-containing 314  benzisoxazole or the isoindole dione moiety played crucial roles in the potency of these 315  kinds of inhibitors, the Se or N atom of which was subjected to nucleophilic attack by the 316  thiol group of Cys and formed the Se-S or N-S bond. 317  318  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 17    DISCUSSION 319  In the present study, we could identify that four clinical-used drugs, i.e., panobinostat, 320  dacinostat, EBS and disulfiram, two anti-cancer drugs, an anti -stroke or -bipolar drugs, 321  and an alcohol-deterrent drug, respectively, could protect the gastric cells from the 322  infection at submicromolar concentrations (Table 1 and Figure 5). The efficacy of these 323  drugs substantially exceeded that of AHA, a well-known urease inhibitor and clinically 324  used drug for bacterial infections. They seemed also to be more effective than tinidazole, 325  a metronidazole type antibiotic in the classic triple recipe for H. pylori (Figures 5). 326  Moreover, panobinostat, EBS and disulfiram have been administered to humans and do 327  not incur severe side effects(28,32,33). Additionally, these drugs did not affect the 328  viability of mammalian cells at a concentration up to 100 M or 25 M (Figure S7B), 329  suggesting that they had a rather safe profile in cells and in vivo. Taken together, our 330  study armed with the newly-developed HTS assay for urease repositions four clinically 331  used drugs as new advanced leads for the treatment of H. pylori infection. 332  The mode of action of panobinostat, dacinostat, EBS or disulfiram was found to inhibit H. 333  pylori urease and reduce the production of NH3 in culture (Table 1; Figures S6A, 4B, 4C 334  and 5C), which are well-known bacterial virulence factors(15). Panobinostat and 335  dacinostat are reversible hydroxamic acid-type inhibitors for urease, and displayed more 336  than 250 or 130 -fold potencies than its analog AHA (Table 1). These largely improved 337  inhibitors indeed enhanced the protective effects to the infection of H. pylori in the 338  cell-based infection model (Figures 5A and 5C), demonstrating that pharmacologically 339  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 18    targeting urease could offer an effective treatment for H. pylori and HPU is a validated 340  pharmacological drug target. However, suppression of the urease activity with these 341  potent inhibitors of HPU, could not retard the growth of H. pylori in culture, indicating 342  that urease is not crucial for bacterial growths. 343  Moreover, EBS was found to irreversibly inhibit urease by covalently modifying an 344  allosteric Cys residue outside of the active site (Figures 2A and 3). The newly identified 345  covalently allosteric regulation of the activity and stability of urease by EBS and captan 346  may explain why these inhibitors could potently and persistently inhibit urease activity 347  and the growth of H. pylori even in the presence of high concentrations of urea substrate 348  (Figure S6B), two merits that are observed for covalent allosteric drugs(34). Indeed, 349  when compared with the reversible inhibitor AHA, EBS displayed an ~ 400 and 10-fold 350  improved potency for JBU and HPU, respectively, and a long-acting inhibitory effect on 351  the endogenous activity of urease and the growth and infection of H. pylori in culture 352  (Figures 4C-D, 5B-C and S6B). Importantly, the anti-H. pylori MIC value of EBS and its 353  analogs, i.e. EBS oxide, 1, 4, 6, seems to be much effective or at least comparable to 354  metronidazole or clarithromycin, which are the two antibiotics in the classic triple recipe 355  for H. pylori (Table S1)(35), indicating these newly-validated chemical moieties for 356  inhibiting the growth of H. pylori are promising antibiotics for developing new 357  treatments for urease-containing pathogens. Since the urease activity is dispensable for 358  the growth of H. pylori (see our discussions with the mode of action of panobinostat and 359  dacinostat), this finding indicates the effect of EBS-type inhibitor on the growth of H. 360  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 19    pylori is beyond the solo inhibition of urease activity. 361  In summary, we identified five clinical drugs as submicromolar inhibitors for plant or 362  bacterial urease by performing the first HTS campaign of urease. These clinically used 363  drugs panobinostat, dacinostat, EBS and disulfiram inhibit the virulence of H. pylori in a 364  gastric-cell-based infection model. This study provides a new HTS assay, drug leads and 365  a regulatory mechanism to develop bioactive urease inhibitors for the treatment of H. 366  pylori infection, especially antibiotic-resistant strains. 367  368  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 20    EXPERIMENTAL PROCEDURES 369  Materials 370  Jack bean urease (JBU), DMSO, and dithiothreitol (DTT) were purchased from Sigma 371  (Steinheim, Germany). Hypochlorous acid, sodium nitroprusside, salicylate, potassium 372  sodium tartrate, urea, sodium hydroxide, bovine serum albumin, Triton X-100, 373  L-histidine and L-cysteine were purchased from Sangon (Shanghai, China). Nessler's 374  reagent was purchased from Jiumu company (Tianjin, China). Acetohydroxamic acid was 375  purchased from Medchemexpress (Monmouth Junction, NJ). Columbia blood agar plate, 376  liquid medium powder for H. pylori, bacteriostatic agent and polymyxin B were 377  purchased from Hopebio company (Shandong, China). RMPI 1640 medium and fetal 378  bovine serum (FBS) were purchased from Gibco (Invitrogen, Gaithersburg, MD). The 379  other materials were purchased from the indicated commercial sources or were from 380  Sigma. 381  Construction of the high-throughput screening assay for urease 382  The assay was constructed to measure the activity of urease based on a 192-tandem 383  microwell plate, which we had previously developed to detect the H2S gas generated by 384  H2S-generating enzymes(24,25). Phosphate or Tris buffer at various pH values were used 385  to determine the optimal pH for JBU in the presence of 25 mM urea substrate (Figure 386  S1E). The optimal conditions were found to be the 50 mM phosphate buffer and pH 7.4. 387  Moreover, the suitable detection reagent and enzyme concentrations were resolved by 388  testing three types of NH3 detection reagents with various concentrations of JBU or HPU, 389  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 21    i.e., salicylic acid-hypochlorite, Nessler’s reagent and phenol red detection reagent 390  (Figures S1A-C). The optimized conditions for the standard assay were found to be with 391  salicylic acid-hypochlorite and commercial Nessler’s detection reagents (Jiumu, Tianjin, 392  China) for JBU and HPU, respectively, in the presence of 50 nM JBU or 200-400 nM 393  HPU, 25 mM urea, 100 M NiCl2, and 50 mM phosphate buffer (final concentrations of 394  pH 7.4). The salicylic acid-hypochlorite detection reagent contained 1.6 mM hypochlorite, 395  400 mM sodium hydroxide, 36 mM salicylic acid, 18 mM potassium sodium tartrate and 396  1.6 mM sodium nitroprusside. The assay was performed using multichannel pipettes to 397  add 1 μl of each compound (solubilized in DMSO or H2O) and 24 μl of the enzyme mix 398  (100 nM, 100 M Tris, pH 7.9) into the reaction well (Figure 1A), followed by a 30-min 399  incubation. After addition of 50 l of salicylic acid-hypochlorite or Nessler’s detection 400  reagent to the detection well, 25 l substrate solution (50 mM urea, 200 M NiCl2, 401  0.04% bovine serum albumin (w/v)) was mixed with the enzyme in the reaction well. The 402  reaction was monitored at 37 °C, and the absorbance at 697 nm or 420 nm was 403  accordingly measured at the appropriate time points in a microplate reader (Synergy2 404  from BioTek, Winooski, VT). 405  Primary screening of urease inhibitors using a high-throughput assay 406  We screened 3,904 compounds of FDA or FAD-approved drugs from Johns Hopkins 407  Clinical Compound Library (JHCCL, Baltimore, MD) or from TopScience Biotech Co. 408  Ltd. (Shanghai, China) at 100 μM for the inhibition of JBU under standard assay 409  conditions with salicylic acid-hypochlorite detection reagent as described above. The Z’ 410  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 22    value of the screening assay was calculated from 60 negative samples (2% DMSO) and 411  60 positive samples (800 M AHA) and found to be more than 0.9 (36), indicating the 412  assay is an excellent assay. Routinely, 16 negative samples and 8 positive samples were 413  used to determine the assay performance, and screening data with a minimum Z’ value of 414  0.5 were accepted. 415  Compounds that show more than 50% inhibition were selected for the further validation. 416  Primary hits were defined as that compound is free of heavy metal atom and shows a 417  more than 50% inhibition at 50 M. 418  Compounds used for follow-up studies 419  All hits identified from the primary screening and their analogs were reordered in the 420  highest pure powder from commercial sources or synthesized in-house for the following 421  studies: dose-dependent, kinetic studies, biophysical assays, LC-MS/MS analysis, cell or 422  bacteria-based studies. Panobinostat and dacinostat were brought from AdooQ (catalog 423  number: A10518 for panobinostat, A10516 for dacinostat). EBS and captan were 424  purchased from Sigma (catalog number: E3520 for EBS, 32054 for captan). Disulfiram 425  (tetraethylthiuram disulfide) was purchased from TCI Chemicals (B0479). Captafol 426  (1ST21228) was purchased from Alta Scientific Co.,Ltd (Tianjing, China), and dibenzyl 427  diselenide (catalog number: B21278) was purchased from Alfa Aesar (Ward Hill, MA). 428  Abexinostat (catalog number: HY-10990), belinostat (HY-10225), vorinostat 429  (HY-10221), ricolinostat (HY-16026), ilomastat (HY-15768) and pracinostat (HY-13322) 430  were brought from Medchemexpress. The purities of these commercially available 431  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 23    primary leads or analogs of leads as well as in-house synthesized EBS derivatives were 432  confirmed to be at least 95% by using HPLC (for details, see below), with an exception 433  for EBS, the purity of which is determined with combustion analysis methods by the 434  supplier. All the HPLC spectra as well as the combustion analysis data for these 435  inhibitors, which were determined either from commercial supplier or by ourself, were 436  included in the Supporting Information (see below). 437  Determination of IC50 values 438  The IC50 values of all the hits or their analogs, as well as AHA, on the activity of JBU, 439  HPU or OAU were determined according to the above-described standard assay 440  conditions. Compounds were incubated with the enzyme and assayed at a series of 441  concentrations (at least 7 steps of doubling dilution). Similarly, the IC50 values of these 442  inhibitors for hCBS or hCSE were determined accordingly(24). Sigmoidal curves were 443  fitted using the standard protocol provided in GraphPad Prism 5 (GraphPad Software, 444  San Diego CA). IC50 was calculated by semilogarithmic graphing of the dose-response 445  curves. 446  Aggregation-based assay 447  To exclude the mechanism by which inhibitors suppress the activity of urease via 448  colloidal aggregation, we performed an aggregation-based assay in the presence of 449  nonionic detergents(37). Freshly prepared Triton X-100 (Sangon, Shanghai, China) at 450  different concentrations of 0.1%, 0.05%, 0.01%, 0.005%, and 0.001% was first tested for 451  its effects on the activity of JBU under standard assay conditions. Subsequently, the 452  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 24    inhibitory effects of panobinostat, dacinostat, EBS, captan and disulfiram, as well as the 453  analogs of EBS in the in vitro JBU activity assay, were determined in the presence of 454  0.01% Triton X-100, a concentration that alone has no inhibitory effect on the activity of 455  JBU. 456  Reversibility assay 457  To illustrate the mode of action for the inhibitors of urease, we performed the 458  rapid-dilution experiment. After incubation with panobinostat at a concentration of 4 M, 459  dacinostat at 10 M, EBS or captan at 200, 100, 50 or 20 μM for 60 min, JBU (10 M) 460  was diluted 200-fold in the assay buffer. After a further incubation of 0, 1, 1.5, 2, 3, 4 or 5 461  h, the remaining activity of JBU was accordingly measured (METHODS). The inhibitor 462  concentrations after dilution are indicated in the figure. 463  Determination of kinact or KI parameters for irreversible inhibitors 464  The IC50 values of EBS or captan for JBU were measured after different preincubation 465  periods with the enzyme, i.e., 5, 10, 20, 30, 40, 45, 60, 70 or 90 min. The kinact and KI 466  values for EBS or captan were obtained by nonlinear regression plotting of the 467  time-dependent IC50 data as previously reported(29). 468  Enzyme kinetics 469  The reaction rate was determined with JBU at the indicated concentrations of panobinosta, 470  dacinostat, EBS or captan against increasing concentrations of urea substrate (15.625, 471  31.25, 62.5, 125, 250, 500, 1000 mM for panobinosta and dacinostat; 12.5, 25, 50, 100, 472  200 mM for EBS and captan). The data were fitting to the Michaelis-Menten inhibition 473  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 25    equation for determination of the competitive and noncompetitive inhibition parameter Ki 474  and Ki using GraphPad Prism 5 (Table 1, Figures 2C and 3B)(24), respectively. To 475  illustrate the inhibition type, Lineweaver-Burk plots of these inhibitors were drawn and 476  analyzed. 477  LC-MS/MS analysis 478  JBU at a concentration of 12.5 M was incubated with DMSO, 200 M EBS or 200 M 479  captan for 120 min at room temperature. Then, three aliquots of 25 g samples from the 480  inhibitor-treated JBU or purified HPU (fraction 3 in Figure 4B) were digested separately 481  with three proteases, including 0.5 l trypsin (1 gl, 0.5 l GluC (1 glor 0.5 l 482  subtilisin (1 glovernight. The proteolytic peptides were combined and desalted on 483  C18 spin columns and dissolved in buffer A (0.1% formic acid in water) for LC-MS/MS 484  analysis. The peptides were separated on a 15-cm C18 reverse-phase column (75 μm × 485  360 μm) at a flow rate of 300 nl/min, with a 75-min linear gradient of buffer B (0.1% 486  formic acid in acetonitrile) from 2% to 60%. The MS/MS analysis was performed on the 487  Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) using 488  standard data acquisition parameters as described previously(38). The mass spectral raw 489  files were searched against the protein database derived from the standard sequence of 490  JBU, HPU or the proteome of H. pylori using Proteome Discovery 1.4 software (Thermo 491  Fisher Scientific, San Jose, CA), with a differential modification of 274.18 m/z in the 492  case of EBS and 150.15 m/z in the case of captan. 493  Surface plasmon resonance assays 494  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 26    The direct interactions between panobinostat, dacinostat, ebselen or captan and JBU were 495  observed by the surface plasmon resonance (SPR) experiment with a BIAcore T200 (GE 496  Healthcare, Uppsala, Sweden). JBU was immobilized on the surface of the CM5 sensor 497  chip via the amino-coupling kit. The working solution used for the SPR assay was PBS-P 498  (10 mM Na2HPO4, 1.8 mM KH2PO4, 2.7 mM KCl, and 140 mM NaCl in presence of 5% 499  DMSO, pH 7.4). To determine the affinity of the inhibitors toward JBU, panobinostat, 500  dacinostat, EBS or captan were diluted to specific concentrations with PBS-P buffer (for 501  panobinostat: 25, 12.5 6.25, 3.125, 1.56 M; dacinostat: 100, 50, 25, 12.5 6.25, 3.125, 502  1.56 M; EBS: 1000, 500, 250, 125, 62.5, or 31.25 nM; for captan: 390.6, 195.3, 97.6, 503  48.8, 24.4, 12.2 or 6.1 nM) and subjected to the JBU-coated chips. The KD values were 504  calculated with BIAcore evaluation software (version 3.1). 505  Molecular modeling 506  The crystal structures of ureases were obtained from the Protein Data Bank (PDB code: 507  4GOA for JBU; PDB code: 1E9Y, HPU). The binding modes of panobinostat or 508  dacinostat were gathered by using the CDOCKER module of the Discovery Studio 509  software (version 3.5; Accelrys, San Diego, CA). Alternatively, AutoDock Vina was 510  initially used to dock the EBS or captan to the respective Cys-containing allosteric site of 511  JBU to obtain the appropriate configurations, enabling the reactive motifs of the 512  compounds (the Se-containing benzisoxazole of EBS and the isoindole dione moiety of 513  captan) to fall into the distance restraint of one covalent bond to the sulfur atom of the 514  reactive Cys residue. The Se-S bond or the N-S bond for isoindole dione was then 515  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 27    manually incorporated using the Discovery Studio 3.5 software (Accelrys, San Diego, 516  CA). Subsequently, molecular dynamics simulation was performed with AMBER14 517  software and the ff03.r1 force field(39). To relieve any steric clash in the solvated system, 518  initial minimization with the frozen macromolecule was performed using 500-step 519  steepest descent minimization and 2,000-step conjugate gradient minimization. Next, the 520  whole system was followed by 1,000-step steepest descent minimization and 19,000-step 521  conjugate gradient minimization. After these minimizations, 400-ps heating and 200-ps 522  equilibration periods were performed in the NVT ensemble at 310 K. Finally, the 100-ns 523  production runs were simulated in the NPT ensemble at 310 K. The binding modes for 524  these inhibitors were visually inspected and the best docking mode was selected. 525  Bacterial strains and culture conditions 526  Bacterial strains of H. pylori or O. anthropic were obtained from BeiNuo Life Science 527  (Shanghai, China). The strains were maintained on Columbia blood agar plates (Hopebio, 528  Shandong, China) containing 5% defibrinated sheep blood at 37 °C under microaerobic 529  conditions (5% O2, 10% CO2 and 85% N2), which was supplied by an 530  AnaeroPack-MicroAero gas generator (Mitsubishi Gas Chemical Company, Japan). After 531  a culture of 3-5 days in the plate, the bacterial colonies were scratched into the liquid 532  medium for H. pylori, containing 10% or 7% fetal bovine serum and an antibacterial 533  cocktail (composed of 10 mg/l nalidixic acid, 3 mg/l vancomycin, 2 mg/l amphotericin B, 534  5 mg/l trimethoprim and 2.5 mg/l polymyxin B sulfate; BeiNuo, Shanghai, China), and 535  microaerobically incubated for another 3 or 5 days. Then, the medium or bacterial cells 536  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 28    were collected for subsequent experiments. 537  A single colony of O. anthropic was inoculated into Luria-Bertani liquid medium (LB), 538  which was supplemented with 50 mg/l ampicillin, 30 mg/l kanamycin and 10% FBS 539  (Invitrogen) and cultured at 37 °C. After the bacterial culture reached an O.D. of 0.8 at 540  600 nm, the bacterial cells were collected by centrifugation for future experiments. 541  The identification of H. pylori and O. anthropic strain was carried out by PCR 542  amplification of the urease gene or 16S rRNA with known primers (Table S4), 543  LC-MS/MS analysis of proteins in the extracts, the bacterial urease activity assay or 544  Gram staining. 545  16S rRNA sequencing 546  One colony from the H. pylori or O. anthropic culture plate was suspended in 50 μl of 547  sterile water, and the DNA was liberated by a boiling-freezing method. The 16S rRNA 548  gene was selectively amplified from this crude lysate by PCR using the universal primers 549  27f and 1492r, which have been previously described (Table S4). The PCR products at 550  ~1400 bp were sequenced. The resultant 16S rRNA sequences were compared with the 551  standard nucleotide sequences deposited in GenBank with the BLAST program 552  (http://www.ncbi.nlm.nih.gov/blast/). The DNA sequences of 16S rRNA extracted from 553  these strains were confirmed to be from H. pylori or O. anthropic. 554  Preparation of crude extracts from the H. pylori and O. anthropic strains for the 555  urease activity assay 556  For the urease activity assay, H. pylori or O. anthropic was cultured accordingly in 100 557  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 29    ml of broth medium as described above. Bacteria were centrifuged at 5,000 rpm for 30 558  min, and the pellet was washed with phosphate-buffered saline (PBS, pH = 7.4). The 559  pellet was resuspended in 7 ml of PBS in the presence of protease inhibitors 560  (Sigma-Aldrich, Steinheim, Germany) and then sonicated for 30 min of 30 cycles (30 s 561  run and 30 s rest) using the noncontact ultrasonic rupture device (Diagenode, Liege, 562  Belgium). The resultant bacterial lysate was centrifuged twice at 12,000 rpm for 30 min; 563  the supernatant was collected and desalted using a Sephadex G-25 desalting column (Yeli, 564  Shanghai, China). The protein in the fractions was separated by 10% SDS-PAGE, and the 565  corresponding protein band for urease was quantified to determine the concentration of 566  ureases by Coomassie blue R-250 (Sinopharm, Shanghai, China) staining using bovine 567  serum albumin as a standard. The desalted fractions were stored at -80 °C in the presence 568  of 15% glycerol until usage in the activity assay. 569  Size-exclusion chromatography for the purification of urease from H. pylori 570  The crude extract from H. pylori was first centrifuged at 12,000 rpm for 30 min. One 571  milliliter of supernatant was loaded onto a gel filtration column (10 mm × 30 cm; GE 572  Healthcare) and eluted with PBS at a rate of 0.5 ml/min on an AKTA Explorer 100 FPLC 573  Workstation (GE Healthcare). The protein peaks observed were collected in Eppendorf 574  tubes in a volume between 0.5 and 1 ml. The collected fractions were separated by PAGE 575  on a 10% Tris-glycine SDS-gel and stained with Coomassie Brilliant Blue R-250 to 576  identify H. pylori urease. 577  Determination of the minimal inhibition concentration and dose-dependent 578  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 30    growth-inhibition curve for urease inhibitors 579  The minimal inhibition concentration (MIC) and dose-dependent growth-inhibition curve 580  for the inhibitors on H. pylori were determined using the broth dilution method(40). 581  Briefly, H. pylori was grown to an OD600 nm of 1.0 in liquid medium supplemented with 582  7% FBS under standard culture conditions. Then, 150 μl H. pylori in the diluted culture 583  (OD of 0.1) was incubated with the inhibitors at final concentrations of 1, 2, 4, 16, 32, 64, 584  128, 256, 512 μg/ml or at indicated concentrations for 72 h. The OD600 nm was measured 585  to calculate the percentage of growth inhibition. The DMSO (1% final 586  concentration)-treated H. pylori cultures and culture medium in the absence of bacteria 587  were referred as the negative control (0%) and positive control (100%), respectively. The 588  MIC was defined as the lowest concentration of inhibitor that inhibited 100% of bacterial 589  growth. The H. pylori strain was found to be resistant to tinidazole or metronidazole and 590  have an MIC of greater than 512 g/ml. 591  Bacterial-cell-based assay for measuring the activity of urease in culture 592  The endogenous activity of HPU in bacterial cultures was determined using the 593  tandem-well-based plate. Briefly, 300 μl of H. pylori culture (OD600 nm ~1.0) was treated 594  with panobinostat, dacinostat or EBS as well as EBS analogs for 6 or 24 h at different 595  concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100 or 200 μM). Then, the bacterial cells 596  were centrifuged, washed and resuspended in assay buffer containing 25 mM urea. 597  Finally, the ~100 l suspension was added to the reaction well of the tandem-well plate 598  and assessed for the activity of urease with Nessler’s reagent under standard assay 599  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 31    conditions. 600  Gastric cell infection model of H. pylori 601  The cell infection model of H. pylori was constructed using the SGC-7901 602  adenocarcinoma gastric cell line and following an established protocol(15). Briefly, H. 603  pylori was cultured in liquid medium for H. pylori at 37 °C for 3-5 days under standard 604  culture conditions (see above). Then, H. pylori at a concentration of 1.5  106 CFU/ml 605  was treated with the indicated inhibitors for 24 h in culture. The bacterial suspension 606  together with 10 mM urea were subsequently added to the culture medium of SGC-7901 607  cells (MOI = 30), which had been cultured with RPMI 1640 medium plus 10% FBS in a 608  96-well plate for one day, and coincubated with the cells for an additional 24 h. Cell 609  images were obtained at specific time points prior to and one day after addition of the 610  bacterial culture using Image Xpress Micro® XLS (Molecular Devices, Sunnyvale, CA) 611  under a 20  objective lens. The cell numbers in the images were quantified using Image 612  Xpress Software. The protective effects of the inhibitors were calculated by dividing the 613  number of SGC-7901 cells after the 24-h treatment by that prior to the treatment (100%) 614  in the same well. 615  616  617  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 32    DATA AVAILABILITY 618  All data are contained within the manuscript. 619  CONFLICT OF INTEREST 620  The authors declare no conflicts of interest. 621  ACKNOWLEDGEMENTS 622  We thank David Sullivan, Jun Liu and Curtis Chong of Johns Hopkins University for 623  providing the Johns Hopkins Clinical Compound Library. We thank Prof. S.C. Tao 624  (Shanghai Center for Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, 625  China) for kindly providing the SGC-7901 cell line. We thank Dr. J.R. Xu (Department 626  of Radiology, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, 627  Shanghai, China) for assisting with the surface plasmon resonance assay experiment. 628  Funding 629  This work was supported by the National Natural Science Foundation of China 630  (31870763, 21834005), the Natural Science Foundation of Shanghai (18ZR1419500), the 631  Shanghai Foundation for the Development of Science and Technology (19JC1413000), 632  and the Research Fund of Medicine and Engineering of Shanghai Jiao Tong University 633  (YG2019QNB27). 634  AUTHOR CONTRIBUTIONS 635  F.L., J.Y., J.Y.X., X.Y.W. and F.W. designed the study, and analyzed the data. F.Z.L. and 636  Y.X.Z. synthesized analogs of EBS lead. Y.Y.Z. constructed the assay and performed the 637  high-throughput screening. H.Q.F. and L.J.L. performed the LC-MS/MS analysis. Q.L. 638  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 33    and Z.P.X. confirmed the inhibitory activity of compounds. S.S.H performed the 639  molecular simulation. F.L., X.Y.W. and F.W. wrote the paper. All authors reviewed the 640  results and approved the final version of the manuscript. 641  642  643  .CC-BY-NC-ND 4.0 International licensemade available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. 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It is The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 37     767  Figure 1 Development of a new high-throughput assay for urease and the discovery of new 768  urease inhibitors. (A) Diagram of the tandem-well-based assay for the NH3-producing enzyme. The 769  procedures for the assays and the cross-section of a tandem-well are shown. Blue, the reaction reagent; 770  red, the detection reagent for NH3. (B) Validation of the urease assay with the known inhibitor AHA. 771  (C) Well-to-well reproducibility of the 192-tandem-well-based assay for urease. ●, 2% DMSO 772  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 38    (control, 100%); ■, 200 μM AHA; ▲, 800 μM AHA (n = 60). (D) High-throughput inhibitor 773  screening for JBU with 192-tandem-well plates. Compound concentration: 100 M. (E-F) 774  Dose-dependent effects of panobinostat, dacinostat, EBS, captan and disulfiram on the activity of JBU 775  (E), human CBS (F) or human CSE (F). Means ± SDS (n = 3). All experiments except the primary 776  screening (D) were independently repeated at least twice, and one representative result is presented. 777  778  779  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 39     780  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 40    Figure 2 Panobinostat, dacinostat, EBS and captan inhibit the activity of JBU. (A) Panobinostat 781  and dacinostat are reversible inhibitors, whereas EBS and captan are covalent inhibitors or 782  slow-binding inhibitors toward JBU. Means ± SDs (n = 3). (B) Effects of the incubation period on the 783  IC50 values of panobinostat and dacinostat toward JBU. Panobinostat and dacinostat were 784  preincubated with JBU for the indicated times before performing the standard assay to analyze their 785  inhibitory effects. Means ± SDs (n = 3). (C) Inhibition of JBU by panobinostat or dacinostat as a 786  function of urea concentration. Ki values for panobinostat and dacinostat, 0.02 μM and 0.07 μM, 787  respectively. Means ± SDs (n=3). (D) Surface plasmon resonance assay analysis of the binding of 788  panobinostat or dacinostat to JBU. KD were calculated using Biacore evaluation software and listed in 789  Table 1. (E) The putative binding mode of panobinostat or dacinostat in the JBU active site. 790  Panobinostat and dacinostat were docked into the JBU crystal structure (PDB code: 4GOA) using the 791  Discovery Studio software. Residues surrounding the inhibitor within a distance of 3.5 Å are shown in 792  gray; and hydrogen bonds are represented as green dotted lines. The experiments were independently 793  repeated at least twice, and one representative result is presented. 794  795  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 41     796  Figure 3 EBS or captan allosterically inhibits the activity of urease by covalently modifying a 797  non-active-site Cys residue. (A) The synergistic inhibitory effects of the combinations of EBS, captan 798  or AHA. A dose-dependent synergistic effect of the combination of EBS at the indicated concentrations 799  with 2 M captan was observed (right panel). Data are presented as percentages of the controls (DMSO 800  and 2 M captan alone in the left panel and right panel, respectively, 100%). Means ± SDs (n=3). (B) 801  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 42    Inhibition of JBU by EBS or captan as a function of the urea concentration. αKi for EBS and captan, 0.8 802  μM and 1.1 μM, respectively. Means ± SDs (n=3). (C) Tandem mass spectrometry analysis of the 803  modification site of EBS and captan on JBU. The Cys modification of EBS and captan on JBU were 804  illustrated in the right panels. (D) Surface plasmon resonance assay analysis of the binding of EBS or 805  captan to JBU. (E) The potential binding modes of EBS and captan in JBU. EBS and captan were 806  modeled into the respective allosteric sites presented in the crystal structure of JBU (PDB code: 4GOA; 807  METHODS). The residues within 3.5 Å surrounding the EBS and captan are shown. Hydrogen bonds 808  are indicated as dashed green lines. The experiments were independently repeated at least twice, and 809  one representative result is presented. 810  811  812  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 43    813  Figure 4 Urease inhibitors suppress bacterial ureases or the growth of urease-containing 814  bacteria. (A) Dose-dependent effects of panobinostat, dacinostat, EBS, captan, disulfiram and AHA 815  on the activity of H. pylori urease (HPU, upper panel) or O. anthropic urease (OAU, lower panel) in 816  vitro. (B) Panobinostat, dacinostat, EBS, captan and disulfiram inhibit the activity of purified HPU 817  from size-exclusion chromatography. Chromatography of the purification is shown in the left panel. 818  The collected fractions (numbers 1-12) of the peaks (left panel), as well as the crude extract (number 819  0), were separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (middle 820  panel). The arrows indicate the peak of H. pylori urease (left panel) or subunit A or B of H. pylori 821  urease (middle panel). The collected sample containing the urease (number 3) was tested to evaluate 822  the inhibitory effects of indicated compounds (right panel). The protein identity of fraction 3 was 823  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 44    analyzed by LC-MS/MS (METHODS and Figure S5). (C) The inhibitory effects of panobinostat, 824  dacinostat and newly synthesized EBS analogs (1, 4 and 6) on the activity of HPU in culture. 825  Inhibitors were incubated with the H. pylori bacteria for 6 h. (D) The effects of panobinostat, 826  dacinostat, EBS and its derivatives on the growth of H. pylori. Mean ± SD (n=3). All experiments 827  were independently repeated at least twice, and one representative result is presented. 828  829  830  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 45    831  Figure 5 Panobinostat, dacinostat and EBS inhibits the virulence of H. pylori in cultured gastric 832  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 46    cells. SGC-7901 cells were infected with HP in the presence of 30 M panobinostat (A), 30 M 833  dacinostat (A), 30 M AHA (A), 20 M EBS (B), 20 M disulfiram or 50 M tinidazole (B) for 24 h 834  before capturing the images in bright field by Image Xpress Micro® XLS (Molecular Devices, 835  Sunnyvale, CA) under a 20 × objective lens. A representative image for each treatment condition is 836  shown (n = 3). Scale bars, 100 m. The cell numbers before treatment (100%) or after 24 h of 837  treatment were quantified. (C) The effects of urease inhibitors on the NH3 amount of the cell culture 838  medium. After the treatment, the amount of NH3 in the cell medium of the corresponding samples was 839  quantified with Nessler’s reagent, and the data are shown as percentages of the control (DMSO, 840  100%). Means ± SDs (n=3). Statistical analyses were performed using the raw data by one-way 841  ANOVA with Bonferroni posttests. n.s., no significance; *, p< 0.05; **, p< 0.01; ***, p < 0.001. All 842  experiments were independently repeated twice, and one representative result is presented. 843  844  845  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 47    846  Figure 6. Structure-activity relationships of panobinostat, dacinostat, EBS and captan. (A) The 847  effects of commercially available analogs of panobinostat and dacinostat, newly synthesized EBS 848  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 48    derivatives and commercially available EBS or captan analogs on the activity of JBU. DMSO, 100%. 849  Mean ± SD (n=3). The experiments were independently repeated at least twice, and one representative 850  result is presented. (B) The illustration charts for the structure-activity relationships of hydroxamic 851  acid analogs, EBS or captan. 852  853  854  855  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ 49    Table 1 Indication, chemical structure, IC50, αKi, or KD values of urease inhibitors. 856  857  858  aFrom the enzyme kinetic study 859  bAssay was performed in 50 mM Tris buffer (pH= 7.4). 860  861  862  863  864  Name Application Structure IC50 (M); JBU IC50 (M); HPU IC50 (M); OAU αKi or Ki (M)a IC50 (M); hCBS IC50 (M); hCSE KD (M) Panobinostat Anticancer N H HN NH O OH 0.2 ± 0.006 0.1 ± 0.01 0.07 ± 0.006 0.02 ± 0.01 > 200.0 > 200.0 8.9 ± 0.4 Dacinostat Anticancer N H N NH O OH HO 1.1 ± 0.005 0.2 ± 0.009 0.1 ± 0.01 0.07 ± 0.02 > 200.0 > 200.0 5.3 ± 0.2 Ebselen Anti-stroke; Anti-bipolar Se N O 0.4 ± 0.07 2.8 ± 0.5 3.0 ± 1.0 0.8 ± 0.2 > 200.0 44.3 ± 1.3 0.089 ± 0.005 Captan Pharmaceutical excipient; Fungicide N O O S CCl3 2.3 ± 0.2 3.4 ± 0.5 5.8 ± 1.6 1.1 ± 0.2 > 200.0 > 200.0 0.096 ± 0.006 Disulfiram Alcohol deterrent CH3 CH3 NS SN H3C H3C S S 38.9 ± 2.7 8.9 ± 1.5 35.0 ± 0.1 - > 200.0 > 200.0 - Acetohydrox amic acid Urinary tract infections NO OH 161.8 ±13.4 33.7 ± 1.0b 25.9 ± 1.2 2.8 ± 0.9 2.1 ± 0.8 > 200.0 > 200.0 - .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S1    Supplementary Information 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  8  aKey Laboratory of Systems Biomedicine (Ministry of Education), Shanghai Center for 9  Systems Biomedicine, Shanghai Jiao Tong University, Shanghai, 200240, China 10  bState Key Laboratory of Microbial Metabolism, Sheng Yushou Center of Cell Biology 11  and Immunology, School of Life Science and Biotechnology, Shanghai Jiao Tong 12  University, Shanghai, 200240, China 13  cSchool of Chemistry & Molecular Engineering, East China University of Science and 14  Technology, Shanghai, 200237, China. 15  dState Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, 16  China Pharmaceutical University, Nanjing, 210009, China 17  eHunan Engineering Laboratory for Analyse and Drugs Development of Ethnomedicine 18  in Wuling Mountains, Jishou University, Hunan, 416000, China 19  fShanghai Key Laboratory of Regulatory Biology, School of Life Sciences, East China 20  Normal University, Shanghai, 200241, China. 21  #These authors contributed equally to this work. 22  *To whom correspondence may be addressed. Emails: fang.wu@sjtu.edu.cn 23  24  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S2    Table of Contents 25  EXPERIMENTAL PROCEDURES .............................................................................. S3 26  Figure S1. Development and optimization of the high-throughput assay for urease. .... S12 27  Figure S2. Validation of on-target inhibition of panobinostat, dacinostat, EBS, captan and 28  disulfiram on JBU. .......................................................................................................... S14 29  Figure S3. The mode of action of panobinostat, dacinostat and disulfiram in vitro . .... S16 30  Figure S4. The mode of action of EBS and captan in vitro. .......................................... S18 31  Figure S5. The identification of HPU from extracts of H. pylori by LC-MS/MS. ........ S20 32  Figure S6. EBS and 1 is a long-acting inhibitor for HPU in culture. ............................. S21 33  Figure S7. The effects of inhibitors on the cell viability of gastric SGC-7901 cells and 34  antibiotic resistance of the H. pylori strain. .................................................................... S22 35  Figure S8. The binding modes of inhibitors in ureases. ................................................. S24 36  Table S1. Chemical structures and IC50 values of EBS or captan analogs for ureases ......... 37  ......................................................................................................................................... S26 38  Table S2. The minimal inhibitory concentration of urease inhibitors or known antibiotics 39  for inhibiting H. pylori and their IC50 values in the in cellulo urease assay ................... S27 40  Table S3. Chemical structures and IC50 values of hydroxamic acid-based analogs for 41  ureases ............................................................................................................................. S28 42  Table S4. Primer sequences. ........................................................................................... S29 43  Reference ....................................................................................................................... S30 44  45  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S3    EXPERIMENTAL PROCEDURES 46  Synthesis of EBS analogs 1-6 47  Compound 1-6 were synthesized according to literature procedure(1-3), as shown in 48  Scheme S1. The chemical reagents and solvents are purchased from commercial sources, 49  and used without further purification, unless stated otherwise. 1H NMR spectra for these 50  compounds were recorded with Bruker 400 spectrometer. The chemical shifts of 1H NMR 51  spectra were referenced to tetramethylsilane (δ 0.00 ppm). 52   53  54  Scheme S1. Synthesis of compounds 1-6. 55  Reagents and conditions: (a) HCl, NaNO2, 0 oC, 0.5 h; (b) Na2Se2, 60 oC, 3 h; (c) SOCl2, 56  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S4    85 oC, 3 h; (d) R2NH2, Et3N, CH2Cl2, rt, 4.5 h; (e) Br2, CH2Cl2, reflux, overnight; (f) 57  Cu(NO3)2.xH2O, Et3N, toluene, reflux. 58  General procedure for synthesis of Compounds 1, 4, 5 and 6 (Route A). 59  The 2-aminobenzoic acid or its derivative was treated with hydrochloric acid (2.50 equiv.) 60  and sodium nitrite (1.06 equiv.) in water (0.7 M) at 0 °C to form the corresponding 61  diazonium salt. Then, the diazonium salt solution was added dropwise to a solution of 62  Na2Se2 (0.87 equiv., fresh prepared from selenium powder and NaBH4 in water) at 0 °C 63  under Argon. The stirring was continued at 60 °C for 3 h. After work-up, crude 64  2,2’-diseleno-dibenzoic acid was obtained. Sequentially, the acid was further converted 65  to 2-(chloroseleno)benzoyl chloride with excess SOCl2 and one drop of DMF at 85 oC for 66  3 h. After the removal of thionyl chloride, the crude compound was obtained, and which 67  was treated with different amines (1.2 equiv.) and Et3N (2.0 equiv.) in CH2Cl2 (0.1 M) 68  under Argon to afford products 1 and 4-6, respectively. Silica gel column 69  chromatography was used to purify these compounds, and their HPLC purity was more 70  than 99%. 71   72  2-Phenyl-6-methoxybenzoisoselen-3-one (1) 73  4-Methoxy-2-aminobenzoic acid and aniline were used to give the compound. 1H NMR 74  (400 MHz, CDCl3): δ 8.01 (d, J = 8.8 Hz, 1H), 7.62 (dd, J = 7.6, 0.8 Hz, 2H), 7.43 (t, J = 75  8.0 Hz, 2H), 7.29-7.24 (m, 1H), 7.11 (d, J = 2.0 Hz, 1H), 7.01 (dd, J = 8.4, 2.0 Hz, 1H), 76  3.92 (s, 3H). MS (m/z): 305.0 [M+H]+. 77  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S5    Benzisoselenol-3-one (4) 78  o-Aminobenzoic acid and ammonia were used to give the product. 1H NMR (400 MHz, 79  d6-DMSO): δ 9.17 (br, 1H), 8.06 (d, J = 8.1 Hz, 1H), 7.81 (dd, J = 8.0, 0.8 Hz, 1H), 7.61 80  (td, J = 7.6, 1.2 Hz, 1H), 7.42 (td, J = 7.6, 0.8 Hz, 1H). MS (m/z): 198.9 [M+H]+. 81  2-Propyl-benzisoselenol-3-one (5) 82  o-Aminobenzoic acid and n-propylamine were used to give the product. 1H NMR (400 83  MHz, CDCl3) δ 8.05 (d, J = 8.0 Hz, 1H), 7.63 (d, J = 7.6 Hz, 1H), 7.58 (td, J = 7.6, 1.2 84  Hz, 1H), 7.45-7.40 (m, 1H), 3.83 (t, J = 7.2 Hz, 2H), 1.76 (hex, J = 7.2 Hz, 2H), 1.00 (t, J 85  = 7.2 Hz, 3H). MS m/z: 242.0 [M+H]+. 86  2-Methylthio-benzisoseleno-3-one (6) 87  o-Aminobenzoic acid and thiourea were used to give the product. 1H NMR (400 MHz, 88  d6-DMSO): δ 10.21 (d, J = 0.8 Hz, 1H), 9.98 (d, J = 1.2 Hz, 1H), 8.00 (d, J = 8.4 Hz, 1H), 89  7.88 (d, J = 8.0 Hz, 1H), 7.71 (td, J = 8.0, 1.2 Hz, 1H), 7.45 (t, J = 7.6 Hz,1H). MS (m/z): 90  240.0 [M-NH3] -. 91  92  Synthesis of compound 2. 93  Compound 2 was prepared according to route B (Scheme S1). 2,2'-Dithiobis-benzoic acid 94  was reacted with bromine in CH2Cl2 under reflux and Argon, and then treated with 95  aniline and Et3N in CH2Cl2 at room temperature. After purified the crude product by 96  column chromatography, compound 2 was obtained. 1H NMR (400 MHz, CDCl3): δ 8.11 97  (d, J = 7.6 Hz, 1H), 7.73-7.69 (m, 2H), 7.68-7.65 (m, 1H), 7.51-7.43 (m, 3H), 7.59 (d, J = 98  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S6    8.0 Hz, 1H), 7.33 (t, J = 7.6 Hz, 1H). MS (m/z): 227.0 [M]+. 99  100  Synthesis of compound 3. 101  Compound 3 was synthesized according to route C (Scheme S1). A Schlenk tube 102  equipped with a stirrer bar was charged with isoindoline-1,3-dione, diphenyliododnium 103  salt (2.05 equiv.) and Cu(NO3)2.xH2O (0.1 equiv.) in dry toluene (0.1 M) under Argon. 104  The mixture was heated to 70 °C, followed by the addition of Et3N (1.5 equiv.). After 105  stirring at 70 °C for 8.5 h (monitoring by TLC), the resulting mixture was continued 106  stirring at room temperature overnight. Then, the mixture was concentrated and the 107  residue was purified by column chromatography. 1H NMR (400 MHz, CDCl3): δ 108  7.99-7.94 (m, 2H), 7.80 (dd, J = 5.6, 3.2 Hz, 2H), 7.55-7.49 (m, 2H), 7.47-7.39 (m, 3H). 109  MS (m/z): 223.1 [M]+. 110  111  HPLC method and purity analysis 112  The purity of compounds 1-5, ebselen oxide or dibenzyl diselenide was analyzed on a 113  Waters sunfire silica column (4.6×250mm; Waters, Milford, MA), which is coupled to a 114  Waters HPLC system (e2695). 3 l compound was injected onto the column and 115  separated by a gradient elution [0 min: 95% phase A (hexane), 5% phase B (isopropyl 116  alcohol); 15 min: 60% phase A (hexane), 40% phase B (isopropyl alcohol)] at a flow rate 117  of 0.7 ml/min under room temperature. 118  Similarly, the purity of compound 6 was resolved on a Waters PHERISORB CN column 119  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S7    (4.6×250mm, Waters). 5 l compound 6 was injected onto the column and analyzed at a 120  flow rate of 0.7 ml/min with an isocratic elution of solvent, which is composed of 75% 121  hexane and 25% isopropyl alcohol. 122  The absorbance of the compounds were monitored at a wavelength of 230 nm, and the 123  corresponding spectra were recorded and analyzed for the determination of the purity. 124  125  The purity of EBS analogs, which were newly synthesized in house (Compound 1-6) 126  or obtained from commercial sources (for Ebselen oxide and dibenzyl diselenide), 127  were analyzed by HPLC (for details, see above). 128  129  Compound 1 130  Determined Purity: > 99%; Retention time: 11.30 min 131  132  133  134  135  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S8    Compound 2 136  Determined Purity: > 99%; Retention time: 7.72 min 137  138  139  140  141  Compound 3 142  Determined Purity: > 99%; Retention time: 6.85 min 143  144  145  146  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S9    147  Compound 4 148  Determined Purity: > 99%; Retention time: 13.55 min 149  150  151  152  153  154  155  156  157  158  Compound 5 159  Determined Purity: > 99%; Retention time: 10.85 min 160  161  162  163  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S10    164  Compound 6 165  Determined Purity: > 97%; Retention time: 7.71 min 166  167  168  169  170  171  172  173  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S11    Ebselen oxide (Cayman) 174  Determined Purity: 95%; Retention time: 8.62 min 175  176  177  178  Ddibenzyl diselenide (Cayman) 179  Determined Purity: > 99%; Retention time: 4.78 min 180  181   182  183  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S12    184  185  Figure S1. Development and optimization of the high-throughput assay for urease. 186  Three types of detection reagents, i.e., salicylic acid-hypochlorite (A), Nessler’s reagent 187  (B), and phenol red (C), were used to detect the released NH3 generated by JBU. The 188  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S13    assay was monitored in the presence of various concentrations of JBU and 25 mM urea. 189  The absorbance (O.D.) values at 697 nm, 420 nm or 435 nm were recorded accordingly. 190  (D) Standard curve of the absorbance of indophenol blue at 697 nm versus the NH4Cl 191  concentration. Various concentrations of NH4Cl were mixed with the detection reagent 192  salicylic acid-hypochlorite before measurement of the absorbance at 697 nm in a 193  microplate reader. (E) The pH profile of the activity of JBU. The 50 mM phosphate 194  buffer (■) was used to maintain the pH between 6 and 8, and 50 mM Tris-HCl (●) was 195  used for pH 7 to 9. JBU was dissolved in the respective buffers and assayed at a final 196  concentration of 50 nM. (F-G) The comparison between salicylic acid-hypochlorite and 197  Nessler’s detection reagent for the detection of HPU activity. The assay was performed to 198  detect the urease activity in the extract from H. pylori with salicylic acid-hypochlorite 199  (left panel) and Nessler’s detection reagent (right panel) in the presence of 25 mM urea. 200  Data are presented as the mean ± SD (n=3). The curves were fitted to the data points with 201  GraphPad Prism 5. All the experiments were independently repeated twice, and one 202  representative result is presented. 203  204  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S14     205  Figure S2. Validation of on-target inhibition of panobinostat, dacinostat, EBS, 206  captan and disulfiram on JBU. (A) NH3 did not interfere with the inhibitors. 5 mM 207  NH3·H2O was incubated with various concentration of panobinostat, dacinostat, EBS, 208  captan or disulfiram in assay buffer. The volatile NH3 was analyzed with salicylic 209  acid-hypochlorite detection reagent (OD697 nm). (B) Triton X-100 did not affect either the 210  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S15    activity of JBU or the inhibition potency of panobinostat, dacinostat, EBS, captan or 211  disulfiram as well as EBS analogs. Various concentrations of Triton X-100 were tested for 212  their effects on the activity of JBU. Additionally, the indicated concentrations of 213  panobinostat, dacinostat, EBS, EBS Oxide, captan, 1, 4, 6 or disulfiram were assayed in the 214  presence or absence of 1/10000 Triton X-100 (v/v) to determine whether their inhibitory 215  mechanisms occurred via colloidal aggregation (METHODS)(4). The results are shown as 216  percentages of the respective control (DMSO or H2O, 100%). Mean ± SD (n=3). All 217  experiments were independently repeated twice, and one representative result is presented. 218  219  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S16    220  Figure S3. The mode of action of panobinostat, dacinostat and disulfiram in vitro. (A) 221  The effect of NiCl2 on the inhibition of JBU by panobinostat or dacinostat. NiCl2 at a 222  concentration of 25, 50 or 100 M was added into the assay that is with the various 223  concentrations of panobinostat or dacinostat under standard assay conditions. (B) Effects 224  of cysteine and histidine on the inhibition of JBU with panobinostat and dacinostat. The 225  assay samples were incubated with the indicated concentrations of panobinostat or 226  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S17    dacinostat in the presence or the absence of 100 M Cys or 100 M His. The results are 227  shown as percentages of the control (DMSO, 100%). (C) Reversibility of the inhibition of 228  JBU by disulfiram. After incubation with JBU at 200, 100 μM for 60 min, disulfiram was 229  diluted 200-fold in assay buffer. The diluted concentrations for disulfiram are 1 μM and 230  0.5 μM, respectively, which do not inhibit JBU (Fig. 1E). After a further incubation for 231  0.5 h, the remaining activity of JBU was measured accordingly (METHODS). And the 232  effect of NiCl2 on the inhibition of JBU by disulfiram was shown on the right panel. The 233  results are shown as percentages of the respective control (DMSO, 100%). Mean ± SD 234  (n=3). All experiments were independently repeated twice, and one representative result is 235  presented. 236  237  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S18    238  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S19    Figure S4. The mode of action of EBS and captan in vitro. (A) Effects of dithiothreitol 239  on the inhibition of JBU caused by EBS and captan. The assay was incubated with 4 M 240  EBS or 10 M captan in the presence or the absence of 5 mM DTT. (B) Effects of 241  cysteine and histidine on the inhibition of JBU by EBS and captan. The samples were 242  incubated with the indicated concentrations of EBS or captan in the presence or absence 243  of 100 M Cys or 100 M His. (C) The effect of NiCl2 on the inhibition of EBS by JBU. 244  NiCl2 at a concentration of 12.5, 25, 50 or 100 M was incubated with the various 245  concentrations of EBS under standard assay conditions. (D) The IC50 values of EBS and 246  captan toward JBU were linearly correlated with the concentrations of JBU. EBS and 247  captan were incubated with various concentrations of JBU, and the IC50 values were 248  determined accordingly. (E) The inhibition constants of KI or kinact for irreversible 249  inhibitors were determined according to the methods described in ref. (5). Means ± SDs 250  (n=3). All experiments were independently repeated at least twice, and one representative 251  result is presented. 252  253  254  255  256  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S20    257  258  Figure S5. The identification of HPU from extracts of H. pylori by LC-MS/MS. 259  Fraction 3 collected by size-exclusion chromatography (Figure 4B) was digested with 260  trypsin, GluCand subtilisin, separated from the C18 reverse-phase column and subjected 261  to analysis with a Thermo Q Exactive Orbitrap (Thermo Fisher Scientific). The peptides 262  in red were identified by LC-MS/MS as subunit A or B of H. pylori. The overall coverage 263  of UreB and UreA identified in the analysis of LC-MS/MS was 80.1% and 76.9%, 264  respectively. 265  266  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S21    267  Figure S6. EBS and 1 is a long-acting inhibitor for HPU in culture. (A) Disulfiram 268  dose-dependently and selectively inhibits the growth of H. pylori. Various concentrations 269  of disulfiram were incubated at 37 °C with H. pylori. (B) The inhibitory effects of EBS 270  and 1 on the activity of HPU in cellulo. EBS, 1 or AHA at a concentration of 100 M 271  were incubated with H. pylori bacteria for 24 h. Additionally, one batch of the treated 272  bacteria was washed, diluted into freshly prepared medium without the addition of the 273  inhibitors, and cultured for an additional 6 h. The in cellulo urease activities from the 274  cultured cells under the two treated-conditions were determined accordingly 275  (METHODS). The results are shown as percentages of the control (DMSO, 100%). Mean 276  ± SD (n=3). All experiments were independently repeated at least twice, and one 277  representative result is presented. 278  279  280  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S22    281  Figure S7. The effects of inhibitors on the cell viability of gastric SGC-7901 cells and 282  antibiotic resistance of the H. pylori strain. (A) The H. pylori strain is resistant to 283  treatment with tinidazole or metronidazole. Various concentrations of tinidazole or 284  metronidazole were incubated at 37 °C with H. pylori for 72 h under standard culture 285  conditions, and the OD at 600 nm was recorded using a spectrophotometer to determine 286  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S23    the cell growth of H. pylori (METHODS). (B) The effects of urease inhibitors on the 287  viability of mammalian cells. SGC-7901 cells were incubated with DMSO, the indicated 288  concentrations of panobinosta, dacinostat, EBS or disulfiram for 24 h in a 96-well plate 289  before measurement of cell viability using the CellTiter96® Aqueous One Solution Cell 290  Proliferation Assay (Promega, Madison, WI). The results are shown as percentages of the 291  control (DMSO, 100%). Means ± SDs (n=3). All experiments were independently 292  repeated at least twice, and one representative result is presented. 293  294  295  296  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S24    297  Figure S8. The binding modes of inhibitors in ureases. (A) The putative binding mode 298  of panobinostat (black) or dacinostat (black) in the HPU active site. Panobinostat and 299  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S25    dacinostat were docked into the HPU crystal structure (PDB code 1E9Y; ref. (6)) using 300  the Discovery Studio software. Residues surrounding the inhibitor within a distance of 301  3.5 Å are shown in gray or in the default atom color. (B) Global view of the binding 302  region of EBS (upper panel) and captan (lower) in JBU. In the modeled EBS or captan 303  and protein complex structure (METHODS and Figure 3E), the protein is shown in black, 304  the key residues (His492 and His519) in the active site of JBU in cyan and the inhibitors 305  as well as its attached Cys residue (Cys313 for EBS, Cys406 for captan; Figure 3E) in red. 306  Hydrogen bonds are represented as green dotted lines. 307  308  309  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S26    Table S1. Chemical structures and IC50 values of EBS or captan analogs for ureases. 310  311  312  Name Structure IC50 (M); HPU IC50 (M); JBU IC50 (M); OAU 1 Se N O O 2.0 ± 0.9 0.3 ± 0.007 7.5 ± 0.6 2   S N O > 10.0 1.0 ± 0.002 4.9 ± 1.1 3     N O O > 10.0 > 10.0 > 10.0 4 Se NH O 1.1 ± 0.08 0.8 ± 0.008 2.2 ± 0.1 5   Se N O > 10.0 1.4 ± 0.03 5.3 ± 0.9 6 Se N O NH2 S 1.3 ± 0.4 0.3 ± 0.04 1.7 ± 0.1 Ebselen Oxide Se N O O   1.5 ± 0.2 0.4 ± 0.005 3.3 ± 0.1 Dibenzyl diselenide   Se Se > 10.0 > 10.0 > 10.0 Captafol   N O O S Cl Cl Cl Cl  > 10.0 8.8 ± 0.2 9.1 ± 1.1 .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S27    Table S2. The minimal inhibitory concentration of urease inhibitors or known 313  antibiotics for inhibiting H. pylori and their IC50 values in the in cellulo urease 314  assay. 315  316  Compound H. pylori (MIC) H. pylori (IC50 values in the in cellulo urease assay; M) g/ml M EBS 4 12.5 5.7 ± 1.3 1 2 6.25 4.7 ± 1.1 4 2 12.5 18.5 ± 1.2 6 4 12.5 21.8 ± 1.0 EBS Oxide 4 12.5 23.2 ± 1.1 Captan 32 100 29.5 ± 1.2 Disulfiram 4 12.5 36.3 ± 1.0 Dibenzyl diselenide > 64 > 200 > 200.0 AHA > 16 > 100 - Tinidazole > 512 > 2000 - Metronidazole > 512 > 3000 - MIC: minimal inhibitory concentration 317  318  319  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S28    Table S3. Chemical structures and IC50 values of hydroxamic acid-based analogs for 320  ureases. 321  322  323  324  Name Structure IC50 (M); HPU IC50 (M); JBU Abexinostat O O N H O HN O OH N 1.3 ± 0.2 1.4 ± 0.3 Belinostat O HN S O O NH HO 3.2 ± 0.2 4.7 ± 0.5 Vorinostat O NH O HN HO 14.0 ± 3.9 4.1 ± 1.9 Ricolinostat N NO NH O HN OH N > 20.0 > 20.0 Ilomastat O NH O HN O HN N H OH > 20.0 > 20.0 Pracinostat O HN OH N N N > 10.0 > 20.0 Hydroxylamine H2N OH  > 20.0 > 20.0 .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S29    Table S4. Primer sequences. 325  No. Primer Usage 1 5'- AGAGTTTGATCCTGGCTCAG-3' 5' primer for 16S rRNA 2 5'- AAGGAGGTGATCCAGCCGCA-3' 3' primer for 16S rRNA 3 5'- ATTAATCATTAGATGTATGGCCCTACTACAGGCG-3' 5' primer for UreB 4 5'- AATATACTCGAGCTAGAAAATGCTAAAGAGTTG-3' 3' primer for UreB 326  327  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/ S30    Reference 328  1. Pacula, A. J., Obieziurska, M., Scianowski, J., Kaczor, K. B., and Antosiewicz, J. (2018) 329  Water-dependent synthesis of biologically active diaryl diselenides. Arkivoc, 153-164 330  2. Ngo, H. X., Shrestha, S. K., Green, K. D., and Garneau-Tsodikova, S. (2016) Development of 331  ebsulfur analogues as potent antibacterials against methicillin-resistant Staphylococcus aureus. 332  Bioorgan Med Chem 24, 6298-6306 333  3. Lucchetti, N., Scalone, M., Fantasia, S., and Muniz, K. (2016) Sterically Congested 334  2,6-Disubstituted Anilines from Direct C-N Bond Formation at an Iodine(III) Center. Angew 335  Chem Int Edit 55, 13335-13339 336  4. Irwin, J. J., and Shoichet, B. K. (2016) Docking Screens for Novel Ligands Conferring New 337  Biology. Journal of Medicinal Chemistry 59, 4103-4120 338  5. Krippendorff, B. F., Neuhaus, R., Lienau, P., Reichel, A., and Huisinga, W. (2009) 339  Mechanism-based inhibition: deriving K(I) and k(inact) directly from time-dependent IC(50) 340  values. Journal of Biomolecular Screening 14, 913-923 341  6. Ha, N. C., Oh, S. T., Sung, J. Y., Cha, K. A., Lee, M. H., and Oh, B. H. (2001) 342  Supramolecular assembly and acid resistance of Helicobacter pylori urease. Nature Structural 343  Biology 8, 505-509 344  345   346  .CC-BY-NC-ND 4.0 International licensemade available under a (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 The copyright holder for this preprintthis version posted January 5, 2021. ; https://doi.org/10.1101/2021.01.05.425432doi: bioRxiv preprint https://doi.org/10.1101/2021.01.05.425432 http://creativecommons.org/licenses/by-nc-nd/4.0/