Additional analysis of structure-property relationship relative to antimicrobial activity also highlighted the potential roles polarity and structural modifications, such as quaternary carbons and lack of an amide carbonyl, may have on permeability across N. gonorrhoeae outer membrane. The Ki values for all analogs were determined by entering the resulting IC50 values, probe Kds, and protein concentrations into the FP Ki equation developed by Nikolovska-Coleska et al49 and are presented in Table 1. Sulfonamides inhibit isozyme IX mediated acidification of hypoxic tumors. MIC values for multiple strains of N. gonorrhoeae in presence and absence of CO2 are presented in Table S4. The trend of alkyl branching that led to reduced antimicrobial activity was again observed for analogs 13 15. Three analogs that possess quaternary carbons (4, 8, and 14) were all less potent in antimicrobial assays than their counterparts that contained reduced alkyl branching. These Kd values were used to select optimal conditions to maximize dynamic range for a competitive FP assay (FITC-AZM and protein concentrations shown in Table S1). Nonetheless, the favorable physicochemical properties of the analogs in this study may translate to reduced risk for triage due to toxicity and/or solubility as they are advanced beyond pre-clinical studies. Therefore, we asked whether acetazolamide, a CA inhibitor, could inhibit intestinal carcinogenesis. Media and reagents were purchased commercially: brucella broth, IsoVitaleX and chocolate II agar (Becton, Dickinson and Company, MD), yeast extract and dextrose (Fisher Bioreagents, NJ), protease peptone and nicotinamide adenine dinucleotide (NAD) (Sigma-Aldrich, MO), hematin, tween 80 and pyridoxal (Chem-Impex International, IL) and phosphate buffered saline (PBS) (Fisher Scientific, MA). government site. Careers, Unable to load your collection due to an error. Analogs 31 33 against N. gonorrhoeae CDC 181 and NgCA Ki values. Carbonic anhydrases (CAs) are a group of ubiquitously expressed metalloenzymes that catalyze the reversible hydration/dehydration of CO 2 /HCO 3. C2527I) This was plated onto an ampicillin agar plate and grown overnight at 37 C. Finally, four analogs containing polar pendant groups (24 27) are presented in red spheres. The research program was partially funded by a Purdue Institute for Drug Discovery Programmatic Grant (M.N.S and D.P.F.) This ultimately led to molecules 20 and 23, which displayed minimum inhibitory concentration values as low as 0.25 g/mL equating to an 8- to 16-fold improvement in anti-gonococcal activity compared to acetazolamide. Moreover, both 20 and 23 also retain favorable drug-like physicochemical properties with molecular weights around 300 Da and low predicted QPLogPo/ws. Bowen VB, Braxton J, Davis DW, Flagg EW, Grey J, Grier L, Harvey A, Kidd S, Kreisel K, Llata E, (2019) Sexually Transmitted Disease Surveillance 2018. Taken together, this study presents a class of anti-gonococcal agents with the potential to be advanced for further evaluation in N. gonorrhoeae infection models. Structure-activity relationship studies for acetazolamide-based carbonic anhydrase inhibitors with activity against Neisseria gonorrhoeae. All fluorescence polarization binding assays were performed in 384-well black bottom plates using an assay buffer consisting of 50 mM Tris, 150 mM NaCl, and 1mM DTT (pH 7.4). Analog 23 and AZM provided a range of MICs from 0.5 g/mL to 4 g/mL, with a lone strain for 23 dropping to 0.25 g/mL. 37 Furthermore, the entry of . The initial Ki value for AZM versus hCA I was 250 nM, equating to 3.3-fold selectivity for NgCA. Future work will include investigation of properties that drive permeability into the bacteria as well as structure-guided design of new inhibitors to improve NgCA activity, reduce hCA activity and limit sites of metabolic liability. Specific analog SAR observations may also support this hypothesis. The UK is the first country to allow OTC access to Sanofi's tadalafil-based erectile dysfunction drug Cialis following a successful switch. Exceptions were also noted for a few analogs containing a tertiary carbon, but these analogs never had MIC values worse than 16 g/mL and reduced antimicrobial potency was not applicable to all tertiary carbon containing analogs. Treatment of metabolic alkalosis. For each cell line, the quantity of viable cells after treatment with each compound was expressed as a percentage of the viability relative to DMSO-treated control cells (average of triplicate wells standard deviation). This trend for in vitro activity was not observed with the cyclohexyl containing matched molecular pair 13 and 14. The .gov means its official. Bacterial carbonic anhydrases as drug targets: toward novel antibiotics? It also reduces the production of cerebrospinal fluid (CSF), which relieves excess fluid pressure around the brain and spinal cord. One of the carbonic anhydrase inhibitors that is sometimes effective against absence seizures. With respect to in vitro NgCA activity the increase in alkyl bulk generally improved inhibition of the enzyme. Meanwhile, substitution of an aromatic phenyl group (16) in place of the cyclohexane provided a two-fold reduction of antimicrobial activity and 7-fold reduction of NgCA activity. MICs of molecules under normal and CO2 conditions. Uses; . Consequently, this rebounding at 24 hours could be attributed to the concentration of 20 potentially becoming diminished below the MIC at that timepoint and suggests frequent dosing for analog 20. Even with this hypothesis we must note that sulfonamide containing drugs used to be a common therapeutic option to treat gonorrhea in the 1930s through mid-1940s and they were determined to inhibit bacterial dihydropteroate synthase (DHPS);62 however, by the late-1940s >90% of gonococcal strains were resistant to sulfonamides.6365 If a second target was potentially contributing to the antimicrobial activity of the molecules presented herein, like DHPS, we would expect analogs to maintain an appreciable level antimicrobial activity in higher CO2 levels, as in the case of azithromycin. Lysed cells were pelleted by centrifuging them at 14000 x g for 1 hour. Modification to the thiazole ring in 32 reduced activity in both NgCA and hCA II by 56-fold compared to AZM, suggesting the change had the same detrimental effect on each CA and that these analogs may interact with NgCA similar to how CAIs bind to hCA II. Acetazolamide is a carbonic anhydrase inhibitor that primarily inhibits . 8600 Rockville Pike N. gonorrhoeae was grown in brucella supplemented broth to logarithmic phase and further diluted to reach an initial inoculum of 6 106 CFU/mL. FDA-approved carbonic anhydrase inhibitors with antimicrobial activity against N. gonorrhoeae. The importance of the carbonyl on the amide for antimicrobial activity was also noted as three analogs (28 30) containing an amine linkage, rather than amide, all exhibited significant reduction of antimicrobial activity with MIC values of > 64 g/mL. Winum, J. Y. et al. MICs (g/mL) of sulfa drugs as compared to CAIs against N. gonorrhoeae strains, Figure S2. An investigation of a set of carbonic anhydrase inhibitors for efficacy against N. gonorrhoeae was carried out. Gonorrhea is a sexually transmitted disease caused by the bacterial pathogen Neisseria gonorrhoeae that colonizes urogenital, anal, and nasopharyngeal tissues. Three of the most potent analogs in terms of antimicrobial activity (21 23) were also among the most potent with regards to Ki against NgCA. We attempted to rationalize this difference in activity by applying the eNTRy rules for Gram-negative Escherichia coli bacteria developed by Richter et al69. Cultures were spun down at 4000 x g for 20 minutes. We then plotted these metrics in a 3-dimensional scatter plot with tPSA on the X-axis, QPLogPo/w on the Y-axis and MIC value on the Z-axis (Figure 7). Concentrations were transformed to log10 based values and the control 1 and 2 (listed above) were normalized to 0 and 100% probe bound to protein, respectively. the contents by NLM or the National Institutes of Health. Important drug interactions with topical carbonic anhydrase inhibitors. Digging deeper, two of these analogs, 32 and 33 (purple spheres), had steep reductions in NgCA activity (Ki > 400 nM) that may explain the reduced antimicrobial activity. This observation suggests alkane cyclization may improve permeability compared to the linear alkanes and will be investigated further. We introduced a 4th dimension in bubble size that corresponds to the Ki values for each analog against NgCA in the CO2 hydration assay. Hewitt et al. For example, when comparing the cyclohexyl containing analog 13 (MIC = 2 g/mL) there was a steep decrease in activity for the quaternary carbon containing nearest analog 14 (MIC > 64 g/mL). Humans express at least 16 -carbonic anhydrase (hCA) isoforms in various tissues.54 Given that these enzymes are ubiquitous throughout the human body, it is critical to gain an understanding for how the new analogs inhibit these enzymes to assess potential drug distribution and toxicity liabilities. Further, in order to investigate the possibility of DHPS inhibition as a target for our analogs we tested the antibacterial activity of sulfamethoxazole, sulfdoxine, and sulfathiazole against three N. gonorrhoeae strains. This translated to an MIC50 value of 0.5 g/mL for 20 against a panel of 30 clinical isolates and it outperformed azithromycin with respect to antimicrobial activity. For human colorectal adenocarcinoma (Caco-2) cell line toxicity the assay went as described previousy.44 Briefly, cells were cultured in Dulbeccos Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), non-essential amino acids (1X), penicillin-streptomycin at 37 C with CO2 (5%). Characterization and purification data for all analogs, as well as the synthetic procedure for FITC-AZM, are provided in the supporting information of this manuscript. activity [20], and in the respiratory response to carbon dioxide [21]. The absorbance values represent an average of three samples analyzed for each compound. Several SAR trends were observed in both MIC and in vitro inhibition assays and at times these SAR trends diverged in opposite directions when comparing antibacterial activity to NgCA potency. Results are presented as percent viable cells relative to DMSO (negative control to determine a baseline measure for the cytotoxic impact of each compound). Drugs and compounds were added and serially diluted along the plates. Carbonic anhydrase fluorescence polarization competition assay data, Table S3. In fact, analog 8 (Ki = 9.1 nM) was among the most potent of the entire study against NgCA but had an MIC of only 16 g/mL; however, within this congeneric series the MIC values did correlate to NgCA potency. In theory, this acute dosing timeline could reduce the impact of any negative side-effects associated with inhibition human carbonic anhydrases. Both molecules displayed the same MIC50 and MIC90 values of 2 and 4 g/mL, respectively. However, it is important to note that these rules were developed in E. coli as the model organism and there likely may be differences in the porins between E. coli and N. gonorrhoeae that could limit the applicability of the eNTRy rules to N. gonorrhoeae. Synthetic route for analogs 1 30. Antibacterial carbonic anhydrase inhibitors: an update on the recent literature, Mancuso F, De Luca L, Angeli A, Berrino E, Del Prete S, Capasso C, Supuran CT, Gitto R, (2020), In silico-guided identification of new potent inhibitors of carbonic anhydrases expressed in Vibrio cholerae, Del Prete S, Nocentini A, Supuran CT, Capasso C, (2020), Bacterial -carbonic anhydrase: a new active class of carbonic anhydrase identified in the genome of the gram-negative bacterium Burkholderia territorii, Angeli A, Ferraroni M, Pinteala M, Maier SS, Simionescu BC, Carta F, Del Prete S, Capasso C, Supuran CT, (2020), Crystal structure of a tetrameric type II -carbonic anhydrase from the pathogenic bacterium Burkholderia pseudomallei, Farha MA, French S, Stokes JM, Brown ED, (2018), Bicarbonate alters bacterial susceptibility to antibiotics by targeting the proton motive force, Inhibition of carbonic anhydrase in neisseria: effects on enzyme activity and growth, Chirica LC, Elleby B, Jonsson B, Lindskog S, (1997), The complete sequence, expression in Escherichia coli, purification and some properties of carbonic anhydrase from Neisseria gonorrhoeae, Huang S, Xue Y, Sauer-Eriksson E, Chirica L, Lindskog S, Jonsson BH, (1998), Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide, Elleby B, Chirica LC, Tu C, Zeppezauer M, Lindskog S, (2001), Characterization of carbonic anhydrase from Neisseria gonorrhoeae, Remmele CW, Xian Y, Albrecht M, Faulstich M, Fraunholz M, Heinrichs E, Dittrich MT, Mller T, Reinhardt R, Rudel T, (2014), Transcriptional landscape and essential genes of Neisseria gonorrhoeae, Kaur J, Cao X, Abutaleb NS, Elkashif A, Graboski AL, Krabill AD, AbdelKhalek AH, An W, Bhardwaj A, Seleem MN, Flaherty DP, (2020), Optimization of acetazolamide-based scaffold as potent inhibitors of vancomycin-resistant enterococcus, The carbon dioxide hydration activity of carbonic anhydrase I. Stop-flow kinetic studies on the native human isoenzymes B and C, Petreni A, De Luca V, Scaloni A, Nocentini A, Capasso C, Supuran CT, (2021), Anion inhibition studies of the Zn(II)-bound -carbonic anhydrase from the Gram-negative bacterium Burkholderia territorii, Analysis of protein-ligand interactions by fluorescence polarization, Elbaum D, Nair SK, Patchan MW, Thompson RB, Christianson DW, (1996), Structure-based design of a sulfonamide probe for fluorescence anisotropy detection of zinc with a carbonic anhydrase-based biosensor, Nikolovska-Coleska Z, Wang R, Fang X, Pan H, Tomita Y, Li P, Roller PP, Krajewski K,, Saito NG, Stuckey JA, Wang S, (2004), Development and optimization of a binding assay for the XIAP BIR3 domain using fluorescence polarization, Nafi BM, Miles RJ, Butler LO, Carter ND, Kelly C, Jeffery S, (1990), Expression of carbonic anhydrase in neisseriae and other heterotrophic bacteria, Alhashimi M, Mayhoub A, Seleem MN, (2019), Repurposing salicylamide for combating multidrug-resistant Neisseria gonorrhoeae, Investigation of auranofin and gold-containing analogues antibacterial activity against multidrug-resistant Neisseria gonorrhoeae, Seong YJ, Alhashimi M, Mayhoub A, Mohammad H, Seleem MN, (2020), Repurposing fenamic acids drugs to combat multidrug-resistant Neisseria gonorrhoeae. However, these changes abolished antimicrobial activity as the polar pendant groups such as pyrrolidine (24), piperidine (25), morpholine (26), and piperazine (27) all had MICs values > 64 g/mL. If this assumption is indeed shown to be true then the variance in antimicrobial potency between the Gram-positive and Gram-negative pathogens may be less due to difference in target inhibition and more so due to differences in accessibility to the intracellular targets. After 24 hours, analogs 20 and 23 reduced N. gonorrhoeae load by 1.9- and 2.6-log10 units, respectively. The analogs did show weak antimicrobial activity (range of 16 32 g/mL) in the CO2 conditions against a few strains (Table S4), however, the difference between ambient and CO2 conditions was still an 8- to 16-fold reduction in potency. Pathogenic N. gonorrhoeae strains are increasingly resistant to common front-line antibiotics. Drugs used in this study were purchased from chemical vendors: acetazolamide (Sigma-Aldrich, MO) and azithromycin (TCI America, OR). (B) Cell viability in human colorectal ladenocarcinoma (Caco-2) cells. Analogs 22 and 23 each exhibited reduced hCA I activity (hCA I Ki = 945 and 855 nM, respectively). This presentation occurs in the elderly, patients with renal or liver failure, people with diabetes, and newborns. However, 14 still maintained NgCA activity (Ki = 75.8 nM) comparable to AZM, and many other analogs, yet the molecule was inactive in antimicrobial assays at concentrations up to 64 g/mL. Molecule 32, in which a nitrogen is replaced with a carbon to provide a thiazole core, displayed an 8.5-fold reduction in NgCA activity compared to AZM with a Ki value of 632 nM. The antimicrobial active sulfonamide analogs in the present work lack a primary amine, or any positive charge for that matter. Plot was made in Excel using the 5dchart add-in (www.5dchart.com). An asterisk (*) indicates a statistically significant difference (P<0.05) between treatment with drugs/compounds compared to DMSO treatment (negative control). This, coupled with improved activity toward NgCA, provided selectivity windows in favor of NgCA that were > 1,300-fold for 22 and 100-fold for 23. Assay plates were then read on a Synergy Neo 2 (BioTek) using FP settings (excitation wavelength 485 nm and emission wavelength of 530 nm) with autogain setting on probe alone control wells (autogain polarization values set to 20 and intensity set to 10,000). 1961 Mar;131:271-4. These observations, along with those discussed prior, suggest that perhaps the quaternary carbon limits molecule permeability into N. gonorrhoeae. Among the linear alkane analogs the tert-butyl representatives, 4 and 8, displayed the most potent Ki values (30.5 and 9.1 nM, respectively) within their respective congeneric series yet were the least potent in antimicrobial assays. This is illustrated by the congeneric series of analogs ranking in order of activity of H (1) = methyl (AZM) > ethyl (2) > iso-propyl (3) > tert-butyl (4) with increased alkyl branching trending to poorer MIC values. The error bars represent standard deviation values for each test agent studied. Acetazolamide (compound 1) was the first non-mercurial diuretic to be used clinically in 1956 . Briefly, cells were cultured in serum-free keratinocyte medium (KSFM) supplemented with 0.05 mg/ml bovine pituitary extract, 0.1 ng/ml epidermal growth factor, and 44.1 mg/liter calcium chloride, at 37 C with CO2 (5%). Yet again, these analogs all displayed NgCA inhibitory activity comparable to AZM and other active analogs. With an increasing occurrence of multidrug-resistant enterococci, there is a need for alternative strategies to treat enterococcal infections. Nonetheless, these studies and ours indicate the presence of a primary amine and avoidance of a negatively charged analog may not be as important for permeability in N. gonorrhoeae. Analog 32 replaced the 4-nitrogen directly flanking the sulfonamide substituent with a carbon resulting in a thiazole scaffold and led to an 8.5-fold reduction of NgCA potency in the CO2 hydration assay and a complete loss of anti-gonococcal activity. 1Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, 575 Stadium Mall Dr., West Lafayette, IN, 47907, USA, 2Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, VA, 24061, USA, 3Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Polo Scientifico, Firenze, Italy, 4Department of Comparative Pathobiology, College of Veterinary Medicine, Purdue University, 625 Harrison St., West Lafayette, IN 47907, USA, 5Purdue Institute for Drug Discovery, 720 Clinic Dr., West Lafayette, IN, 47907, USA, 6Purdue Institute of Inflammation, Immunology and Infectious Disease, 207 South Martin Jischke Dr., West Lafayette, IN, 47907, USA, Figure S1. 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