We also screened a collection of organomercurial compounds (Figure 49). These compounds were analyzed in response to the discovery of the inhibitory ability of mersalyl acid. The compounds selected for screening were phenyl mercuric borate (70), methyl mercuric chloride (71), mersalyl acid (72), ethylmercurylthiosalicylic acid (73), phenyl mercuric acetate (74), p- acetoxymercurianiline (75), o-chloromercuryl phenol (76), phenylmercuric salicylate (77), thiomersal (78), merbromin (79), and p-chloromercuribenzoic acid (80). Compounds were initially analyzed at 100 μM concentration in duplicate (Table 4) and those indicating high levels of inhibition (70-77, 80% inhibition or higher) were rescreened at 1 μM in triplicate. The percent inhibition at 1 μM is given in Table 5.
118
Figure 49. Structures of organomercurials analyzed for Prp inhibitory activity: phenylmercuric borate (70), methylmercuric chloride (71), mersalyl acid (72), ethylmercurylthiosalicylic acid (73), phenylmercuric acetate (74), p-acetoxymercurianiline (75), o-chloromercuryl phenol (76), phenylmercuric salicylate (77), thiomersal (78), merbromin (79), p-chloromercuribenzoic acid (80).
119
Table 4. Calculated inhibitory levels of 70-80. Compounds were screened in triplicate, so standard deviation cannot be calculated. 70-77 were rescreened at 1 μM but 78-80 were abandoned due to lack of inhibition at 100 μM.
Table 5. Inhibition data for compounds 70-77, which appear to inhibit Prp. Compounds were screened at 1 μM and DMSO concentration was normalized.
Compound % Inhibition at 1 μM 70 71 ± 17 71 64 ±7 Compound (100 μM) Slope % Inhib. 70 Trial 1 -52 101 70 Trial 2 -33 100 71 Trial 1 -101 101 71 Trial 2 -60 101 72 Trial 1 -69 101 72 Trial 2 -46 101 73 Trial 1 -56 101 73 Trial 2 -19 100 74 Trial 1 89 99 74 Trial 2 170 98 75 Trial 1 210 96 75 Trial 2 172 97 76 Trial 1 158 98 76 Trial 2 36 100 77 Trial 1 1397 81 77 Trial 2 1523 79 78 Trial 1 7452 -1 78 Trial 2 7212 3 79 Trial 1 7014 6 79 Trial 2 6905 7 80 Trial 1 7062 5 80 Trial 2 7459 -1
120 72 59 ± 5 73 36 ± 1 74 29 ± 6 75 28 ± 2 76 16 ± 2 77 8 ± 3
Given the unusual inhibitory profile of Prp to traditional broad-spectrum protease inhibitors, we pursued other compounds capable of inhibition. Mercury is thiophilic,433 and we suspected that the active-site cysteine of Prp could be inhibited by certain commercially available organomercurials. Mersalyl is an organomercurial that has been used therapeutically as a diuretic434,435 and served as a positive control in the development of our HTS assay. Phenylmercuric borate (Merfen orange),436,437 mersalyl,434,435 phenylmercuric acetate,438,439 thiomersal,440,441 and merbromin (Mercurochrome)437,442 have been used for their antiseptic properties in topical ointments. Phenylmercuric acetate is used as an antimicrobial in eyedrops and is used to combat ocular fungal pathogens in addition to its herbicidal functionality.439 Thiomersal (also known as thimerosal) was a preservative in vaccines meant to prevent fungal growth within the vials.440,441 The resulting controversy caused by thiomersal’s mercury content has become a popular topic for groups interested in adverse effects caused by vaccination. In recent redeeming research, the toxicity of thiomersal is being investigated for its potential as a cancer treatment.434,443–445 Thiomersal is capable of inducing apoptosis in multiple cancer cell types, including prostate,443 gastric,444 and oral cancers.445 This indicates the opportunity for further research into mercury-containing compounds as a strategy to target harmful cell types using their innate toxicity. Our research contributes to this trend by revealing the potential for organomercurial compounds as antibiotics and inhibitors of Prp.
121
3.5 Conclusions
As a novel, never-drugged, essential enzyme for Firmicutes, Prp has high potential as an antibiotic target. Research into inhibiting Prp indicates that the active site cannot be targeted by known broad-spectrum protease inhibitors. This specificity notwithstanding, we have demonstrated that Prp can be inhibited by small-molecule organomercurial compounds. Because of the toxicity related to organomercurials, though, these compounds may not behave well as therapeutics, so other options must be explored. Given the specificity of the binding site of Prp, a rationally designed suicide inhibitor based on the L27 extension may be a promising project for the future.
122
REFERENCES
(1) Kola, M.; Urba, K. Antibiotic selective pressure and development of bacterial resistance.
Int. J. Antimicrob. Agents 2001, 17, 357–363.
(2) Keyser, P.; Elofsson, M.; Rosell, S.; Wolf-Watz, H. Virulence blockers as alternatives to antibiotics: type III secretion inhibitors against Gram-negative bacteria. J. Intern. Med. 2008, 264, 17–29.
(3) Gaytan, M. O.; Martinez-Santos, V. I.; Soto, E.; Gonzalez-Pedrajo, B. Type three secretion system in attaching and effacing pathogens. Front. Cell. Infect. Microbiol. 2016, 6, 129.
(4) Tsai, C. L.; Burkinshaw, B. J.; Strynadka, N. C. J.; Tainer, J. A. The Salmonella type III secretion system virulence effector forms a new hexameric chaperone assembly for export of effector/chaperone complexes. J. Bacteriol. 2015, 197, 672–675.
(5) Duncan, M. C.; Wong, R.; Dupzyk, A. J.; Bray, W. M.; Linington, R. G. Inhibitors of the
Yersinia pseudotuberculosis type III secretion system. Antimicrob. Agents Chemother. 2014, 58,
1118–1126.
(6) Bailey, L.; Gylfe, Å.; Sundin, C.; Muschiol, S.; Elofsson, M.; Nordström, P.; Henriques- Normark, B.; Lugert, R.; Waldenström, A.; Wolf-Watz, H.; et al. Small molecule inhibitors of type III secretion in Yersinia block the Chlamydia pneumoniae infection cycle. FEBS Lett. 2007, 581, 587–595.
(7) Collmer, A.; Badel, J. L.; Charkowski, A. O.; Deng, W. L.; Fouts, D. E.; Ramos, A. R.; Rehm, A. H.; Anderson, D. M.; Schneewind, O.; van Dijk, K.; et al. Pseudomonas syringae Hrp type III secretion system and effector proteins. Proc. Natl. Acad. Sci. U. S. A. 2000, 97, 8770– 8777.
123
(9) Clements, A.; Young, J. C.; Constantinou, N.; Frankel, G. Infection strategies of enteric pathogenic Escherichia coli. Gut Microbes 2012, 3, 71–87.
(10) Deng, W.; Li, Y.; Vallance, B. A.; Finlay, B. B. Locus of enterocyte effacement from
Citrobacter rodentium: sequence analysis and evidence for horizontal transfer among attaching
and effacing pathogens. Am. Soc. Microbiol. 2001, 69, 6323–6335.
(11) Elliott, S. J.; Wainwright, L. A.; McDaniel, T. K.; Jarvis, K. G.; Deng, Y. K.; Lai, L. C.; McNamara, B. P.; Donnenberg, M. S.; Kaper, J. B. The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol. Microbiol. 1998, 28, 1–4.
(12) Muschiol, S.; Bailey, L.; Gylfe, Å.; Sundin, C.; Hultenby, K.; Bergström, S.; Elofsson, M.; Wolf-Watz, H.; Normark, S.; Henriques-Normark, B. A small-molecule inhibitor of type III secretion inhibits different stages of the infectious cycle of Chlamydia trachomatis. Proc. Natl.
Acad. Sci. U. S. A. 2006, 103, 14566–14571.
(13) Kühne, S. A.; Hawes, W. S.; La Ragione, R. M.; Woodward, M. J.; Whitelam, G. C.; Gough, K. C. Isolation of recombinant antibodies against EspA and intimin of Escherichia coli O157:H7. J. Clin. Microbiol. 2004, 42, 2966–2976.
(14) Cardenal-Muñoz, E.; Ramos-Morales, F. Analysis of the expression, secretion and translocation of the Salmonella enterica type III secretion system effector SteA. PLoS One 2011,
6, e26930.
(15) Munera, D.; Crepin, V. F.; Marches, O.; Frankel, G. N-terminal type III secretion signal of enteropathogenic Escherichia coli translocator proteins. J. Bacteriol. 2010, 192, 3534–3539. (16) Yang, J.; Hocking, D. M.; Cheng, C.; Dogovski, C.; Perugini, M. A.; Holien, J. K.; Parker, M. W.; Hartland, E. L.; Tauschek, M.; Robins-Browne, R. M. Disarming bacterial virulence
124
through chemical inhibition of the DNA binding domain of an AraC-like transcriptional activator protein. J. Biol. Chem. 2013, 288, 31115–31126.
(17) Kimura, K.; Iwatsuki, M.; Nagai, T.; Matsumoto, A.; Takahashi, Y.; Shiomi, K.; Omura, S.; Abe, A. A small-molecule inhibitor of the bacterial type III secretion system protects against
in vivo infection with Citrobacter rodentium. J. Antibiot. (Tokyo). 2011, 64, 197–203.
(18) Khebizi, N.; Boudjella, H.; Bijani, C.; Bouras, N.; Klenk, H. P.; Pont, F.; Mathieu, F.; Sabaou, N. Oligomycins A and E, major bioactive secondary metabolites produced by
Streptomyces sp. strain HG29 isolated from a Saharan soil. Journal de Mycologie Medicale. 2018,
150–160.
(19) Cundliffe, E.; Thompson, J. The mode of action of nosiheptide (Multhiomycin) and the mechanism of resistance in the producing organism. J. Gen. Microbiol. 1981, 126, 185–192. (20) Morgan, J. M.; Duncan, M. C.; Johnson, K. S.; Diepold, A.; Lam, H.; Dupzyk, A. J.; Martin, L. R.; Wong, R.; Armitage, J. P.; Linington, R. G. Piericidin A1 bocks Yersinia Ysc type III secretion system needle assembly. Am. Soc. Microbiol. 2017, 2, e00030-17.
(21) Lee, J. H.; Regmi, S. C.; Kim, J. A.; Cho, M. H.; Yun, H.; Lee, C. S.; Lee, J. Apple flavonoid phloretin inhibits Escherichia coli O157:H7 biofilm formation and ameliorates colon inflammation in rats. Infect. Immun. 2011, 79, 4819–4827.
(22) Chang, C. Y.; Krishnan, T.; Wang, H.; Chen, Y.; Yin, W. F.; Chong, Y. M.; Tan, L. Y.; Chong, T. M.; Chan, K. G. Non-antibiotic quorum sensing inhibitors acting against N-acyl homoserine lactone synthase as druggable target. Sci. Rep. 2014, 4, 7245.
(23) Huber, B.; Eberl, L.; Feucht, W.; Polster, J. Influence of polyphenols on bacterial biofilm formation and quorum-sensing. Zeitschrift fur Naturforsch. 2003, 58, 879–884.
125
(24) McHugh, R. E.; O’Boyle, N.; Connoly, J. P. R.; Hoskisson, P. A.; Roe, A. J. Characterization of the mode of action of aurodox, a type III secretion system inhibitor from
Streptomyces goldiniensis. Infect. Immun. 2019, 87, e00595-18.
(25) Chinali, G. Synthetic analogs of aurodox and kirromycin active on elongation factor Tu from Escherichia coli. J. Antibiot. (Tokyo). 1981, 34, 1039–1045.
(26) Nakasone, N.; Higa, N.; Toma, C.; Ogura, Y.; Suzuki, T.; Yamashiro, T. Epigallocatechin gallate inhibits the type III secretion system of Gram-negative enteropathogenic bacteria under model conditions. Fed. Eur. Microbiol. Soc. Lett. 2017, 364, fnx111.
(27) Bélanger, L.; Garenaux, A.; Harel, J.; Boulianne, M.; Nadeau, E.; Dozois, C.M.
Escherichia coli from animal reservoirs as a potential source of human extraintestinal pathogenic