Structure-Activity Relationship Studies for 1,3,4-Thiadiazole-Based Bacterial Carbonic Anhydrase Inhibitors with In Vivo Efficacy against Drug-Resistant Neisseria gonorrhoeae.

Structure-Activity Relationship Studies for 1,3,4-Thiadiazole-Based Bacterial Carbonic Anhydrase Inhibitors with In Vivo Efficacy against Drug-Resistant Neisseria gonorrhoeae - PMC Skip to main content An official website of the United States government Here's how you know Here's how you know Search Log in Primary site navigation Search Log in Search PMC Full-Text Archive Search in PMC Journal List User Guide As a library, NLM provides access to scientific literature. Inclusion in an NLM database does not imply endorsement of, or agreement with, the contents by NLM or the National Institutes of Health. Learn more: PMC Disclaimer | PMC Copyright Notice J Med Chem . 2026 May 6;69(10):12183–12204. doi: 10.1021/acs.jmedchem.6c00012 Structure-Activity Relationship Studies for 1,3,4-Thiadiazole-Based Bacterial Carbonic Anhydrase Inhibitors with In Vivo Efficacy against Drug-Resistant Neisseria gonorrhoeae Prabhakara R Tharra † , Molly S Youse † , Nader S Abutaleb ‡, § , Abdallah S Abdelsattar ‡, § , Katrina J Holly † , Alessio Nocentini ∥ , Ahmed A Abouelkhair ‡, § , Carolyn K Metcalfe † , Chao-Jan Liao ⊥, # , Venkatesh P Thriumalaikumar ⊥, # , Farman Ali † , Anil Kumar Marapaka † , Faith L Drummond † , Olivia C Snell † , Claudiu T Supuran ∥ , Mohamed N Seleem ‡, §, * , Daniel P Flaherty †, ∇, ○, * Author information Article notes Copyright and License information † Borch Department of Medicinal Chemistry and Molecular Pharmacology, College of Pharmacy, Purdue University, West Lafayette, Indiana 47907, United States ‡ Department of Biomedical Sciences and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States § Center for One Health Research, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, United States ∥ Department of NEUROFARBA, Section of Pharmaceutical and Nutraceutical Sciences, University of Florence, Sesto Fiorentino 50019, Italy ⊥ Purdue Proteomics Facility, Bindley Bioscience Center, Purdue University, West Lafayette, Indiana 47907, United States # Center for Plant Biology, Department of Botany and Plant Pathology, Purdue University, West Lafayette, Indiana 47907, United States ∇ Purdue Institute for Drug Discovery, West Lafayette, Indiana 47907, United States ○ Purdue Institute of Inflammation, Immunology and Infectious Disease, West Lafayette, Indiana 47907, United States * Email: seleem@vt.edu . * Email: dflaher@purdue.edu . Accepted 2026 Apr 29; Collection date 2026 May 28. © 2026 The Authors. Published by American Chemical Society This article is licensed under CC-BY-NC-ND 4.0 PMC Copyright notice PMCID: PMC13224167  PMID: 42089225 Abstract Drug-resistant Neisseria gonorrheae is becoming an increasingly concerning threat as cases continue to rise, highlighting the need for novel therapeutics. We, therefore, have further developed our previously established structure–activity relationship for derivatives of acetazolamide, an FDA-approved carbonic anhydrase inhibitor. A large cohort of acetazolamide-based analogs was investigated for their activity against N. gonorrheae and their binding to NgCAs. Lead compounds were carried forward for in vitro ADME evaluation before prioritizing two molecules, 18 and 33 , for in vivo PK analysis. These compounds were orally bioavailable and metabolically stable, and exhibited low cytotoxicity against mammalian cell lines. When investigated for in vivo efficacy in an infected mouse model, 18 was found to significantly decrease gonococcal bioburden compared with the vehicle control. Our studies culminated in the finding that acetazolamide-based compounds can be developed as effective drugs against N. gonorrheae and may be an answer in the search for new therapeutics. Introduction Gonorrhea is a sexually transmitted infection caused by Neisseria gonorrheae , a Gram-negative bacterium that was estimated by the World Health Organization (WHO) to infect greater than 82 million people worldwide in 2020. Cases of gonorrhea often go unreported, however, due to the asymptomatic infections experienced with rectal or pharyngeal gonorrhea, along with asymptomatic cases of urogenital gonorrhea in women. − If left untreated, complications of the infection include pelvic inflammatory disease, ectopic pregnancies, infertility, chronic pain, and epididymitis. − N. gonorrheae has a history of acquiring resistance to drug classes over time, starting in the 1940s with the sulfonamides, resulting in shifting treatment regimens for gonococcal infection over time. , The recommended treatment by the Centers for Disease Control and Prevention (CDC) is currently a single intramuscular injection of ceftriaxone, though strains have already become less susceptible to the drug, leaving clinicians with a dwindling toolbox to combat this superbug. − Recent advancements in the drug discovery pipeline have been made with two novel bacterial topoisomerase inhibitors, zoliflodacin , and gepotidacin, which were recently approved by the U.S. Food and Drug Administration (FDA) for the treatment of uncomplicated urogenital gonorrhea in adults and children 12 years and older. Though the approval of both agents brings hope for future gonococcal treatments, the history of resistance still underscores the need to explore novel targets to treat gonorrhea. Our efforts to develop novel antibacterial therapeutics have focused on inhibitors for the carbonic anhydrase enzymes (CAs) in N. gonorrhoeae − and Enterococcus species. − CAs are a family of Zn 2+ -metalloenzymes that catalyze the reaction between water and carbon dioxide to produce bicarbonate and a proton. Bicarbonate is a critical carbon source for central carbon metabolism and anaplerotic reactions. , CAs are ubiquitous enzymes, present in all kingdoms of life and categorized into eight distinct familiesα, β, γ, δ, ζ, η, θ, and ι based on their tertiary structures, enzymatic activities, and the organisms in which they are expressed. − Among CA isoforms, humans only encode for the α-CAs class, while N. gonorrhoeae encodes for two isoforms: an α- and β-CA. The α-CA from N. gonorrhoeae (α-NgCA) is essential and is presumed to be localized in the periplasm. , Additionally, apo- and ligand-bound crystal structures have been published, demonstrating ligand-binding interactions with CA inhibitors. , The β-NgCA is less studied in terms of pharmacologic inhibition. This isoform is hypothesized to be a cytosolic enzyme and was reported to be a mediator of CO 2 growth dependence for N. gonorrhoeae since strains carrying a single hypomorphic mutation to β-NgCA require supplemental CO 2 to survive and grow. Given this prior literature and our own studies, targeting the bacterial CAs in N. gonorrhoeae is a viable treatment strategy against this pathogen. , Our team has reported two FDA-approved human CA inhibitors having activity against N. gonorrhoeae . These molecules have been used for decades as diuretics and to treat other conditions such as epilepsy, , glaucoma, − and edema, among other indications. Our team demonstrated that acetazolamide (AZM) ( Figure ) is able to reduce the gonococcal burden in mice. A subsequent report showed that ethoxzolamide (EZM) analogs with improved pharmacokinetic properties displayed oral efficacy to reduce gonococcal burden in mice. Finally, the initial structure–activity relationship (SAR) for the AZM scaffold for inhibition against α-NgCA and antibacterial activity was explored with three analogs, 1 – 3 ( Figure ), exhibiting minimum inhibitory concentration (MIC) values of 0.8–3.1 μM against N. gonorrhoeae isolates, respectively, while demonstrating biochemical inhibition of the N. gonorrhoeae α- and β-CAs. The prior SAR was informative and served as a starting point for continued development. 1. Open in a new tab FDA-approved carbonic anhydrase inhibitor AZM and previously reported analogs with improved antimicrobial activity. MICs against N. gonorrhoeae strain CDC 181. While these initial results were promising, gaps in our understanding of targeting the bacterial CAs in N. gonorrhoeae remain. First, even though in vitro inhibition of the targets has been demonstrated, on-target engagement in cells has yet to be shown. Second, even though a proof-of-concept high-dose study demonstrated in vivo efficacy after oral dose with AZM (50 mg/kg), there have been no efficacy studies performed with optimized AZM analogs. Therefore, in this work, we sought to expand the breadth of AZM-analogs based on our findings from the initial SAR study. The analogs were tested against two strains of N. gonorrhoeae as well as the α- and β-NgCAs to better understand how the AZM scaffold can be further optimized to improve its properties as a CA inhibitor and anti-gonococcal agent. Chemoproteomics experiments were employed to evaluate target engagement in cell lysates for the first time. Lead compounds were evaluated for their in vitro absorption, distribution, metabolism, and excretion (ADME) properties and in vivo pharmacokinetic (PK) evaluation in mice. The study culminates in the evaluation of leads in a gonococcal infection mouse model and assessing the clearance of N. gonorrhoeae bioburden against a ceftriaxone-resistant N. gonorrhoeae strain. Results Chemistry Our initial antigonococcal evaluation of the AZM scaffold identified lead compounds with cyclic hydrophobic groups combined with one or two methylenes in the linker from the amide group. Thus, we continued exploring the SAR by evaluating a cohort of molecules featuring additional modifications to the acetamide moiety of AZM. Either AZM or the commercially available thiazole-containing counterpart N -(5-sulfamoyl-1,3-thiazol-2-yl)­acetamide was used as the initial starting material for the synthesis of all analogs. Analogs were mostly synthesized according to previously reported synthetic routes, and many were recently reported in a SAR study against Enterococcus. Briefly, analogs 5 – 57 were synthesized by first cleaving the acetamide group from AZM or N-(5-sulfamoyl-1,3-thiazol-2-yl)­acetamide under refluxing acidic conditions, affording the free amine intermediates 4a and 4b ( Scheme ). Amide couplings with various acyl chlorides, either purchased from commercial vendors or generated in situ from their respective carboxylic acids, were performed in acetonitrile with base (either triethylamine or potassium carbonate) to generate the analogs 5 – 57 ( Scheme ). To assess how the carbonyl in the AZM scaffold influences antimicrobial activity and CA binding, analog 58 was designed as a direct comparator to compound 2 . This was synthesized via reductive amination of 4a with 2-cyclohexylacetaldehyde and sodium cyanoborohydride ( Scheme ). Analogs with piperidine-based R-groups, 59 – 67 , were synthesized by generating the chloroacetamide intermediate 4c from 4a , followed by coupling to secondary amines with triethylamine in THF ( Scheme ). Due to the availability of starting materials, the carboxylic acid for analog 68 was synthesized by benzyl deprotection via catalytic hydrogenolysis with Pd/C to provide intermediate 4d , followed by generating the acyl chloride in situ and coupling it with 4a ( Scheme ). Lastly, amide coupling of 4a with N-Boc-protected carboxylic acids using triethylamine and EDC generated the intermediates 4e and 4f , which were subsequently deprotected in acidic conditions to afford 69 – 70 , isolated as TFA salts ( Scheme ). 1. Synthesis of Analogs 5 – 57 . Open in a new tab 1 Reagents and Conditions: (a) Concentrated HCl, 95̊C, 16 h, 73–75%; (b) R-COOH, oxalyl chloride, DMF, DCM, 0 °C to rt; (c) R-COOH, thionyl chloride, 60 °C, 2.5 h; (d) acyl chloride (from previous step or purchased commercially), triethylamine, acetonitrile, 0 °C to rt, 16 h; (e) acyl chloride (from previous step or purchased commercially), potassium carbonate, acetonitrile, 0 °C to rt, 16 h, 1–94% over two steps. 2. Synthesis of 58 . Open in a new tab 2 Reagents and Conditions: (a) 2-cyclohexylacetaldehyde, acetic acid, DCM, methanol, rt, 1 h; then sodium cyanoborohydride, DCM, methanol, rt, 16 h, 36%. 3. Synthesis of 59 – 68 . Open in a new tab 3 Reagents and Conditions: (a) 2-Chloroacetyl chloride, TEA, acetonitrile, 0 °C to rt, 16 h, 14%; (b) R-NH, TEA, THF, 0 °C, then 20–60 °C, 16–72 h, 7 – 99%; (c) Pd/C, MeOH, H 2 , 1 h, 99%. (d) 4a, TEA, acetonitrile, 0 °C to rt, 16 h, 22%. Full structures of analogs provided in Table 4. Synthesis of 69 and 70 . Open in a new tab 4 Reagents and Conditions: (a) R-COOH, triethylamine, EDC, acetonitrile, rt, 16 h, 9–12%; (b) TFA, water, DCM, 0 °C to rt, 4 h. Compounds isolated as TFA salts, 90–97%. Anti-Gonococcal Structure–Activity Relationship Analogs were assessed for activity against two strains of drug-resistant N. gonorrhoeae : CDC 178 (tetracycline-, penicillin-, and ciprofloxacin-resistant strain) and CDC 181 (azithromycin-resistant strain) to inform the SAR. The first cohort of analogs was designed based on compound 1, featuring a phenyl pendant group separated from the amide carbonyl by two methylenes ( Table ). A methyl walk was performed on three sets of related analogs with mixed results in terms of potency versus N. gonorrhoeae . The analogs with the methyl-phenyl pendant and a single methylene ( 6 – 8 ) were the most potent of this group (MICs = 1.6–3.2 μM). Shortening or extending the linker, combined with methyl substitution on the phenyl ( 9–14 ) all exhibited reduced anti-gonococcal activity, upwards of 15-fold, with the lone exception being the meta -phenyl analog 10 with no methylene linker with an MIC of 1.6 μM. Finally, placement of a 4-fluoro substituent on analog 15 also led to reduced activity. In summary, the initial SAR evaluation yielded no appreciable improvement in activity over analog 1 . 1. Minimum Inhibitory Concentrations for Compounds 1 and 5–15 . Open in a new tab The second cohort of analogs was based on the cyclohexane pendant derivatives 2 and 3 ( Table ). This set of molecules exhibited a larger range of activity to inform SAR. With respect to methyl-containing analogs, molecules containing single methyl substituents to yield tertiary carbons were generally tolerated and, in some cases, displayed improved potency over the non-substituted cyclohexane derivatives. The most active analogs of this group were the 3-CH 3 analog 18 , which was a mixture of cis and trans diastereomers containing a single methylene linker (MIC = 0.79 μM), and the matched molecular paired analog 33, possessing the same 3-CH 3 in a mixture of cis and trans diastereomers and lacking a linker methylene (MIC = 0.82 μM) against both strains. Starting materials were available to probe the role of the cis - versus trans -isomers on activity in analogs 34 and 35 . The racemic cis -isomer 34 was essentially equipotent to the mixture of diastereomers, while the racemic trans- isomer was about 4–8-fold less potent. Reduced anti-gonococcal potency with increasing alkyl bulk was a trend observed in our prior work, and this trend continues in this study. Increased globularity of the pendant group in the form of a norbornane moiety ( 27 and 28 ) was essentially equipotent to the cyclohexane counterparts (MICs = 1.6–6.1 μM), but incorporating a much larger adamantane system, 29 and 30 , significantly reduced activity with MICs of 22 and 190 μM, respectively. Another interesting SAR comparison came in the form of the methyl-cyclopropyl 31 compared to the bicyclo[1.1.1]­pentane derivative 32, in which the latter structure had an MIC > 100 μM, while the former displayed an MIC value of 7.6 μM. Moreover, analogs that introduced quaternary carbon centers by the addition of methyl groups ( 16 , 21 , 22 , and 31 ) all exhibited reduced potency with MICs of 7.6–190 μM against N. gonorrhoeae . Finally, our prior reports indicated the cyclohexane ring is susceptible to metabolism in mouse liver microsomes; therefore, four fluorinated analogs designed to block potential metabolic hot spots were synthesized to assess the impact on antimicrobial activity. The substitutions to the 4-position of the cyclohexane ( 23 and 24 ) or on the methylene linker ( 25 and 26 ) did not improve or maintain activity and ranged in MICs from 2.9 to 90 μM. 2. Minimum Inhibitory Concentrations for Compounds 2, 3 , and 16–37 a . Open in a new tab We next evaluated a series of spirocycles with the hypothesis that creating alkyl bulk while reducing the flexibility of the scaffold may improve potency. We observed varying effects on activity from this set, with MICs ranging from 1.7 to 220 μM ( Table , analogs 38 – 46 ). Inserting an oxygen into the spirocycle negatively impacted antimicrobial activity. Across three matched molecular pairs 43 compared to 44 and 45, as well as 38 compared to 46 the analogs containing an oxygen within the ring system were 4- to 16-fold less active against N. gonorrhoeae . The insertion of an oxygen was also evaluated in matched molecular pairs of analog 3, in which pyran derivatives 47 and 48 were 2- to 4-fold less potent. Likewise, substituting the cyclobutane moiety in analog 49 with an oxetane ( 50 ) reduced potency by >4-fold. Inserting an oxygen into the methylene linker provided varying effects but appeared to be better tolerated than placement in the alkyl ring system. It is difficult to deduce an SAR due to the different MICs for analogs 3 and 23 against both strains. The insertion of the ether linker ( 51 and 52 ) was essentially equipotent compared to the methylene linker counterparts. A more pronounced effect of the ether is seen in the t -butyl analogs 53 and 54 , where the ether results in a 4- to 8-fold decrease in activity. 3. Minimum Inhibitory Concentrations for Compounds 38–58 a . Open in a new tab We also designed two analog sets to evaluate the role of the nitrogen adjacent to the sulfonamide and the role of the carbonyl in the amide toward antimicrobial activity. In each case, the thiazole heterocycle ( 55 , 56 , and 57 ) resulted in a loss of activity when compared to the thiadiazole counterparts ( 1 , 2 , and 3 ). Thus, the nitrogen appears to be integral to maintaining antimicrobial activity. Additionally, the carbonyl also contributes to antimicrobial activity, as eliminating the carbonyl from the amide in 2, resulting in an amine linker in 58, led to a 64-fold decrease in potency. Prior reported SAR for CA inhibitors against N. gonorrhoeae indicated that the insertion of nitrogen into the alkyl ring was detrimental to antimicrobial activity much like oxygen, as observed above. We sought to investigate nitrogen-containing heterocycles with increased lipophilicity or the addition of fluorine atoms to modulate p K a ( 59 – 68 , Table ). Overall, the analogs were found to result in poor efficacy, with the majority resulting in MICs ≥150 μM. However, there was a trend in SAR observed for the fluorinated piperidine analogs, which correlated with improved activity. We previously reported that the unsubstituted piperidine analog was inactive against N. gonorrhoeae similar to 59 – 62 . The predicted p K a of the protonated piperidine nitrogen in the ring is 9.2, as calculated using Epik (Schrödinger Release 2024–2). Moving from a single 4-F in derivative 66 (predicted nitrogen p K a = 8.66; MIC ≥ 99 μM) to the 4,4-di-F, 67 (p K a = 8.07), and 3,3-di-F, 68 (p K a =7.39), derivatives exhibited improvement in MIC values to 12–23 μM against the two strains tested. The correlation between reducing p K a of the nitrogen indicates a potential role of the protonated nitrogen with respect to activity versus N. gonorrhoeae . 4. Antimicrobial Data for Compounds 59–72 . Open in a new tab Finally, because N. gonorrhoeae is a Gram-negative pathogen, we sought to apply a primary amine to the scaffold to assess if the eNTRy rules for Escherichia coli developed by the Hergenrother laboratory might also apply to N. gonorrhoeae . Thus, a set of matched molecular pairs with primary amine analogs ( 69 and 70 ) were designed to evaluate the effect of this eNTRy rule in the context of N. gonorrhoeae and were compared to alkyl chain analogs previously reported with activity against N. gonorrhoeae ( 71 and 72 ). Interestingly, the primary amine-containing analogs had significantly reduced activity (MIC ≥ 200 μM) compared to the alkyl chain analogs (MIC ranging from 6.8–32 μM). These results suggest that the primary amine does not aid in facilitating entry into N. gonorrhoeae , although further accumulation validation is necessary to confirm this hypothesis. All analogs were also tested for antimicrobial activity against N. gonorrhoeae in 5% CO 2 conditions to confirm if CO 2 supplementation modulates potency. CAs convert CO 2 to bicarbonate; however, this reaction can also occur spontaneously at elevated levels of CO 2 . In the 5% CO 2 environment, the CO 2 traverses the bacterial cell membrane and, within the cell, and can spontaneously convert to bicarbonate, thus supplementing the cell and bypassing the need for a CA. , If the antimicrobial effects of analogs are mediated through inhibition of the CAs, the bacteria will therefore be less susceptible to the compounds. A shift in susceptibility was observed for all analogs tested, where the activity against N. gonorrhoeae in normal ambient air conditions is completely diminished in elevated levels of CO 2 , resulting in MICs >64 μg/mL for all analogs ( Table S1 ). On the other hand, ceftriaxone retained potent activity against the pathogen in the presence of a 5% CO 2 atmosphere because it mediates its mechanism of action by binding to penicillin-binding proteins, not through inhibiting the NgCAs. Carbonic Anhydrase Inhibition Through the course of the project, SAR was primarily informed by the anti-gonococcal activity obtained through susceptibility testing. In our prior work, the biochemical inhibition of analogs against the proposed targets, α-NgCA and β-NgCA, did not correlate with MIC values. However, it is not uncommon for antibiotics to have a disconnect between MIC data and biochemical potency for a few reasons including differences in compound permeability across Gram-negative membranes and potential polypharmacology of molecules. Nonetheless, all analogs were evaluated against the gonococcal CA targets to further understand the changes in AZM analog SAR toward the bacterial CAs. Additionally, analogs were assessed for activity against two of the highest expressed human isoforms, hCA I and hCA II, to evaluate selectivity. For these studies, compounds were subjected to a CO 2 hydration activity assay to determine the inhibition constants ( K i ). In general, analogs exhibited more potent activity against α-NgCA compared to β-NgCA ( Table ). This is expected, as the AZM scaffold was originally developed against human CAs represented by only the β-CA family. While both families utilize Zn 2+ to carry out the hydration reaction, the α-CA family is structurally distinct from the α-CA isoforms. For example, β-CAs are a dimeric catalytic unit with differences in binding site architecture compared to the monomeric unit of α-CAs. To date, there are no ligand-bound β-CA structures; thus, there is a lack of understanding as to the binding interactions with β-CAs beyond Zn 2+ coordination by the sulfonamide moiety. 5. Inhibition Constants ( K i )­ Against N. gonorrhoeae and Human CAs. Cpd α-NgCA β-NgCA hCAI hCAII Cpd α-NgCA β-NgCA hCAI hCAII Cpd α-NgCA β-NgCA hCAI hCAII 1 8.3 60.2 855 8.1 26 29.3 613 35.2 15.0 50 2.80 503 201 24.6 2 36.6 750 58.7 24.5 27 45.8 628 48.0 21.5 51 22.0 82.4 412 7.90 3 0.70 150 946 0.32 28 59.1 458 128 34.1 52 1.90 65.3 333 2.80 5 27.5 169 63.0 51.0 29 61.8 803 72.3 46.9 53 24.8 424 267 10.2 6 29.3 130 51.8 25.7 30 189 251 195 92.8 54 38.4 530 155 26.5 7 24.3 152 42.4 36.8 31 12.9 210 269 39.3 55 82.7 994 1740 58.5 8 33.9 200 91.5 82.3 32 35.8 186 108 30.5 56 462 558 2430 98.7 9 64.2 82.3 105 25.6 33 16.7 316 82.7 31.5 57 724 1440 1580 54.3 10 43.9 66.2 267 13.2 34 12.5 116 55.1 18.3 58 65.7 426 933 38.3 11 34.0 119 344 21.1 35 19.7 104 63.9 14.9 59 43.5 164 44.5 41.6 12 8.31 41.6 886 4.90 36 7.80 239 1520 8.80 60 61.8 198 34.4 59.7 13 10.6 53.4 724 11.8 37 6.90 597 1380 11.8 61 67.6 88.3 54.1 31.1 14 2.10 33.2 613 8.80 38 32.6 452 103 27.3 62 68.0 96.7 59.8 26.1 15 5.70 45.9 696 3.60 39 18.1 399 339 32.6 63 45.1 437 145 43.7 16 58.2 600 153 33.1 40 23.5 375 313 59.4 64 34.3 668 27.5 13.4 17 42.0 628 101 29.2 41 17.3 363 171 33.2 65 22.7 705 91.5 36.8 18 35.4 660 60.4 5.3 42 38.2 322 276 21.7 66 17.1 99.2 68.2 14.1 19 42.9 881 69.2 22.3 43 56.6 204 220 40.1 67 9.9 42.5 57.7 4.30 20 33.6 450 47.6 18.3 44 49.3 173 186 64.0 68 20.1 38.6 51.3 6.90 21 30.1 993 74.4 25.5 45 57.9 170 165 50.8 69 69.4 62.7 189 85.2 22 0.80 169 669 5.3 46 22.5 250 226 24.3 70 45.9 32.5 124 48.9 23 15.1 72.9 55.9 12.4 47 0.75 113 750 0.84 71 27.5 303 213 22.3 24 0.55 103 678 0.48 48 3.40 179 585 3.20 72 8.5 552 328 22.7 25 6.80 95.0 550 4.90 49 16.0 555 334 29.6 Open in a new tab a CO 2 catalytic hydration stopped-flow assay K i values determined from the mean ( n = 3) IC 50 values entered into the Cheng–Prusoff equation and reported in nM. b Data previously reported in ref . Overall, as with our prior report, the trends for the K i values did not always correlate with antimicrobial activity (). However, a few nearest-neighbor analog comparisons against the NgCA targets may provide insight into whether the changes in antimicrobial potency were driven by CA inhibition or perhaps molecule entry into the bacterial cell. For example, comparing 2 to 16 and 21 , in which the only difference is quaternary carbons in the latter two analogs, the K i values against both NgCA isoforms are comparable for all three analogs ( Table ). However, the antigonococcal activity for 2 is approximately 30-fold more potent than that of both 16 and 21 . The same trends in terms of NgCA and MIC activity are observed when comparing 3 to the geminal dimethyl-matched molecular paired analog 22 . On the basis of these data, and those from other pairs, we hypothesize that the reduced MIC activity may be due to decreased access to the intracellular NgCA targets for the quaternary carbon-containing molecules. Similarly, the disconnect in MIC and NgCA values for the alkyl chain derivatives ( 71 and 72 ) compared to the primary amine analogs ( 69 and 70 ) also suggests a lack of intracellular accumulation for the primary amine analogs. The primary amine analogs are 2–5-fold less potent than the corresponding alkyl-matched molecular pair against α-NgCA, yet they are inactive against the bacterial pathogens, accounting for >32-fold less antimicrobial activity. Moreover, analogs with nitrogen insertion into the cyclic alkane rings ( 59 – 68) all exhibit comparable or better K i values against the NgCAs compared to the cyclohexane-containing derivative 2, yet they are significantly less potent against the pathogen. Interestingly, the geminal di-fluoro analogs 67 and 68 begin to make modest gains in both NgCA activity and anti-gonococcal potency, suggesting that the pK a modulation of the tertiary amine may influence both properties. These results further suggest a role of molecule accumulation in the SAR evaluation for these analogs. One set of analogs that the NgCA K i values may at least partially explain the lack of antibacterial potency is for the thiazole heterocycles 55 – 57 . The analogs were among the least active against both NgCAs in the entire set, with 57 exhibiting K i values versus α-NgCA and β-NgCA of 723 nM and 1,440 nM, respectively. Prior reports note that sulfonamide p K a , and deprotonation specifically, is a critical driver of binding to Zn 2+ in CA enzymes. The thiadiazole core of AZM provides the sulfonamide proton on the nitrogen with a p K a value of 7.2 Removing the 4-nitrogen in the thiadiazole to yield a thiazole (analog 55 – 57 ) modulates the pK a of the sulfonamide, with a predicted value of 8.5 using Epik. This modification would reduce the amount of deprotonated molecule in solution at pH 7.4, in turn reducing binding affinity to the Zn 2+ in CAs. While the reduced binding to the putative targets is likely the cause of the reduced antibacterial activity, we have yet to establish if these changes also alter molecule accumulation into the cell. This will be investigated in future work. With respect to activity against hCA I and II, the K i values for the compounds against hCA II were almost always more potent than those against hCA I. Additionally, the potency trends for analogs generally aligned well for hCA II and β-NgCA, whereas there was wide variability in hCA I activity ( Table ). This is likely due to a slight difference within the hCA active sites, primarily in that hCA II has Thr200 with a side chain that interacts with the ligand via hydrogen bonding, while hCA I has a histidine in the corresponding location that can contribute π–π stacking interactions. This difference in active site residues likely explains the molecular selectivity toward hCA II over hCA I. Moreover, β-NgCA has a threonine at the same location as in hCA II, resulting in the K i trends for the analog set more closely aligning between these two enzymes. Activity versus a Panel of N. gonorrhoeae Clinical Isolates With drug resistance being a primary concern in drug discovery against N. gonorrhoeae we sought to evaluate the potencies of lead compounds 18 and 33 against a panel of 17 multidrug-resistant N. gonorrhoeae clinical isolates ( Table ). Values for AZM, azithromycin (AZI), and the current recommended treatment ceftriaxone (CEF) are all provided for comparison. Analog 18 exhibited MIC 50 (the concentration inhibiting 50% of the tested isolates) and MIC 90 (the concentration inhibiting 90% of the tested isolates) values of 0.20 μM and 0.79 μM, respectively. This was followed by 33, with MIC values of 0.82 μM and 1.6 μM. Both significantly improved in the anti-gonococcal spectra compared to AZM and AZI. While CEF remained the most broadly potent against most isolates, analog 18 was 4–8-fold more potent against the CEF-resistant N. gonorrhoeae strains (WHO-X and WHO-Z). 6. MIC Values versus a Panel of N. gonorrhoeae Clinical Isolates. MICs (μM) Strainz 18 33 AZM AZI CEF CDC 166 0.79 1.6 18 1.3 0.097 CDC 167 0.79 1.6 18 5.3 0.012 CDC 173 0.39 1.6 9.0 1.3 0.048 CDC 177 0.10 0.82 9.0 1.3 0.012 CDC 182 0.39 1.6 18 2.7 0.016 CDC 188 0.10 0.82 9.0 1.3 0.048 CDC 202 0.20 0.82 9.0 21 0.012 SPL-4 0.10 0.41 4.5 0.67 0.024 WHO-F 0.39 1.6 9.0 0.33 ≤0.006 WHO-K 1.6 3.3 9.0 0.67 0.048 WHO-M 0.20 0.82 4.5 0.33 0.048 WHO-P 0.20 0.82 9.0 5.3 ≤0.006 WHO-U 0.20 0.82 9.0 5.3 ≤0.006 WHO-V 0.20 0.82 9.0 >170 0.048 WHO-W 0.20 0.82 9.0 0.33 0.048 WHO-X 0.20 0.82 9.0 0.67 1.5 WHO-Z 0.39 0.82 9.0 0.67 1.5 MIC 50 0.20 0.82 9.0 1.3 0.048 MIC 90 0.79 1.6 18 21 0.097 Open in a new tab a MICs performed in standard conditions with incubation in ambient air. When incubated in the presence of 5% CO 2 , all analogs exhibited MICs > 64 μg/mL. MICs are the average of three biological replicates. In Vitro ADME Evaluation of Lead Analogs Our previous study of AZM analogs focused on exploring SAR against N. gonorrheae , and though it provided a foundation by identifying molecules that exhibited strong antimicrobial effects in vitro , we did not investigate the drug-like properties of the compounds. Toward the goal of selecting a molecule able to demonstrate oral efficacy in a mouse model for gonococcal infection, we evaluated analogs 18 and 33 for in vitro ADME properties. Lipophilicity was evaluated by measuring the LogD 7.4 values via the shake-flask method in PBS and 1-octanol. Following general lipophilicity trends, analogs 33 (LogD 7.4 = 1.69) and 18 (LogD 7.4 = 2.26) maintained LogD values in drug-like physicochemical space for oral absorption ( Table ). , 7. In Vitro ADME Properties for Lead Analogs. Solubility (μM) Caco-2 P app Cpd LogD 7.4 PBS SGF SIF MLM (%) A → B B → A ER Mouse Plasma f u Plasma Stability (%) 18 2.26 75 50 45 87 7.7 14.3 1.86 0.13 86 33 1.69 90 60 249 94 6.7 13.1 1.96 0.15 100 Open in a new tab a Bidirectional Caco-2 permeability (× 10 –6 cm/s) (n = 2): A = apical, B = basolateral; pH 6.5 for donor and pH 7.4 for receiver chambers. b Aqueous solubility in PBS at pH 7.4 with 0.05% DMSO v/v. c Simulated gastric fluid, pH = 1.2. d Simulated intestinal fluid, pH = 6.8. e Efflux ratio, B→A/A→B. f f u = fraction unbound in mouse plasma. g Percent molecule remaining in mouse plasma at the 3-h time point. The solubilities of 18 and 33 were evaluated in phosphate-buffered saline (PBS, pH 7.4, 0.05% DMSO v/v), simulated gastric fluid (SGF, pH 1.2), and simulated intestinal fluid (SIF, pH 6.8). In general, a benchmark solubility threshold for drug candidates is typically greater than 10 μM in aqueous media. The lead molecules were soluble in PBS at concentrations of 75 μM and 90 μM for 18 and 33 , respectively ( Table ). Drugs must be solubilized in order to be absorbed through the intestinal lining, and the pH where drug absorption takes place in the small intestine is critical toward optimal drug absorption. Therefore, solubility was evaluated in both SGF (pH 1.2) and SIF (pH 6.8) to ensure that they will remain dissolved in physiological environments necessary for absorption after oral dosing. The compounds’ solubilities ranged from 45 μM to 249 μM across both media. Analogs were then evaluated for stability in the mouse liver microsomal (MLM) assay to investigate metabolic stability. Liver microsomes contain high concentrations of the enzymes largely responsible for Phase I drug transformations, including cytochrome P450 enzymes, esterases, and amidases. The MLM data revealed that the leads had little propensity to undergo Phase I metabolism in the liver, with 87% of 18 remaining and 94% of 33 remaining after 1 h ( Table ). A bidirectional human colon adenocarcinoma (Caco-2) permeability assay is a surrogate for predicting intestinal absorption. Apparent permeability rates (P app ) in both the apical-to-basolateral direction (A→B) and the basolateral-to-apical direction (B→A) with pH 6.5 in the donor chamber and pH 7.4 in the receiver chamber, emulate the intestine-blood pH gradient and were assessed to aid in predicting oral bioavailability. − Analogs 18 and 33 exhibited P app A→B = 7.7 × 10 –6 cm/s and 6.7 × 10 –6 cm/s with P app B→A = 14.3 × 10 –6 cm/s and 13.1 × 10 –6 cm/s, respectively. These values indicate efflux ratios (ER, P app B→A /P app A→B ) < 2, indicating sufficient intestinal absorption with low propensity for efflux. , Next, analogs were evaluated for fraction unbound (f u ) in mouse plasma as an initial indication of the potential for protein binding that would be encountered in vivo. Analogs 18 and 33 exhibited f u values of 0.13 and 0.15, respectively ( Table ), which were higher than AZM’s f u value of 0.05–0.1. 437552 For comparison, the f u for ceftriaxone has been reported to range from 0.06–0.1 in healthy patient plasma, although this unbound fraction increases in critically ill patients and patients with impaired renal function. Lastly, the compounds’ stability in plasma was determined prior to taking the compounds forward for in vivo PK assessment. Compound instability in plasma leads to rapid clearance and short half-lives in vivo and can lead to inaccuracies in in vivo PK studies, as the compound will continue degrading in samples prior to quantification. We therefore thought it was important to confirm plasma stability before moving to in vivo studies. Analogs 18 and 33 were found to be stable in plasma, with 86% and 100% of compound remaining after three hours. Taking all this data into account, the ADME properties of analogs 18 and 33 are favorable for candidates that are being developed for oral dosing. Mammalian Cell Viability Following the encouraging in vitro ADME data for 18 and 33 , the analogs were further investigated for cytotoxic effects in an MTS assay using two mammalian cell lines. AZM is subject to renal elimination and, as a diuretic, it affects the kidneys through bicarbonate diuresis due to the high abundance of CAs present in the kidneys. , We therefore chose to evaluate the analogs for cytotoxicity in monkey kidney fibroblast (Vero) cells. Additionally, human endocervical (ME-180) cells, a common cell line used to study N. gonorrhoeae pathogenesis, were also used in the assay to evaluate the susceptibility of cells commonly affected by the pathogen. Compounds were tested at 100, 200, and 400 μM for 24 hours, representing a >100-fold the MICs against N. gonorrhoeae ( Figure ). A modest reduction in cell viability was observed at 400 μM concentrations for each molecule against ME-180 endocervical cells, resulting in approximately 70–75% cell viability for both analogs. An additional dose-response was performed across a broader range of concentrations, and neither analog demonstrated reduced cell viability below 50% at the highest concentration of 170 μg/mL (>500 μM, ), which would equate to a safety index (CC 50 /MIC 90 ) of >632 for 18 and >312 for 33 . Neither analog exhibited any measurable reduction of cell viability in either cell line at concentrations up to 200 μM, which was >250- and 150-fold the MIC 90 ’s for 18 and 33 , respectively. 2. Open in a new tab Cell viability for 18 ( A ) and 33 ( B ) tested at three concentrations against Vero cells (blue) and ME-180 cells (red), measured by the MTS assay. DMSO-treated cells served as a negative control. Absorbance values were calculated as the average of three biological replicates, and error bars represent the standard deviation from the mean. Antibacterial Killing Kinetics AZM has previously been investigated for its antibacterial killing kinetics in comparison to other anti-gonococcal agents and was found to be bacteriostatic, exhibiting a significant reduction in N. gonorrhoeae FA1090 load compared to DMSO-treated culture after 24 hours. , Leads 1 and 2 from our initial SAR campaign also exhibited bacteriostatic effects against the same strain, reducing bacterial burden by 1.9- and 2.6-log 10 units, respectively, after 24 h. The present leads, 18 and 33 , along with AZM, were assessed at 5 × MIC in triplicate for their anti-gonococcal killing kinetics against N. gonorrhoeae WHO-X strain ( Figure ). Similar to what was observed for prior AZM-based analogs, AZM and 33 each exhibited bacteriostatic effects against N. gonorrhoeae WHO-X with AZM providing a 1.0 Log 10 CFU/mL reduction and 33 yielding a 2.6 Log 10 CFU/mL reduction over 24 hours. Analog 18 demonstrated bactericidal effects with ∼4.0 Log 10 CFU/mL reduction in N. gonorrhoeae burden over the same time frame. These values were all significantly different compared to DMSO-treated samples, which exhibited a 1.2 Log 10 CFU/mL increase in N. gonorrhoeae WHO-X over the same time period. Azithromycin, in agreement with previous reports, demonstrated bactericidal activity, completely clearing the bacterial load (below the limit of detection) after 8 hours. ,,, 3. Open in a new tab Time-kill kinetics for 18 , 33 , and AZM against N. gonorrhoeae WHO-X strain. DMSO (vehicle) served as the negative control, while AZI served as the positive control. All molecules were dosed at 5× MIC. Time points are the mean of three biological replicates, and error bars represent the standard deviation from the mean. The data were analyzed by two-way ANOVA with post-hoc Dunnett’s test for multiple comparisons. * Indicates a statistically significant difference ( P < 0.5) compared to the negative control. The dotted line represents the limit of detection, and the dashed line represents a 3-Log 10 CFU decrease from the initial inoculum. Frequency of Spontaneous Resistance Reports have predicted a future of untreatable gonorrhea due to the worldwide increase in resistance to currently available treatments. Additionally, extensively drug-resistant strains of N. gonorrhoeae that are resistant to ceftriaxone, penicillin, tetracycline, fluoroquinolones, and macrolides have already been identified, compromising the management of gonorrhea cases. , With drug resistance being a primary concern in treating the infection, we investigated whether N. gonorrhoeae may develop resistance toward these molecules. A frequency of spontaneous single-step resistance assay was implemented, dosing N. gonorrhoeae WHO-X strain with analogs 18 and 33 along with AZM at 5× and 10× MIC ( Table ). AZI and rifampicin were used as controls. Consistent with our prior reports for bacterial CA inhibitors, no colonies were isolated at concentrations of 5× or 10× MIC for any of the AZM-based analogs, AZM, nor for AZI. In addition, we tested the frequency of mutation for analogs 18 and 33 at lower concentrations (2× and 3× MIC) to assess the likelihood of development of resistance under lower selective pressure. For the low concentrations, we isolated a few colonies on the plates (3–7 colonies) with analogs 18 and 33 and AZM, suggesting the apparent frequency at 2× and 3× MIC was higher than that at 5× and 10× MIC. However, we tested the susceptibility of all colonies isolated alongside the parent strain, and we did not find any shift in the analogs’ 18 and 33 and AZM MICs against all the isolated colonies, likely reflecting the survival of partially susceptible or tolerant cells rather than stable resistant mutants. Therefore, the frequency of mutation for analogs 18 and 33 and AZM (at 2× and 3× MIC) was considered <7.4 × 10 –9 ( Table ), similar to that of AZI. In contrast, the colonies isolated with rifampicin displayed a high shift in the MICs, indicating resistance was obtained for rifampicin (at 2×, 3×, 5×, and 10× MIC), with a high frequency of mutation (1.6 × 10 –8 –3.6 × 10 –8 ). These results indicate resistance would be difficult to form against the CA inhibitors. 8. Spontaneous Resistance Frequencies of Lead Analogs and AZM Compared to Other Anti-Gonococcal Drugs Against N. gonorrhoeae WHO-X. Drug 2× MIC 3× MIC 5× MIC 10× MIC 18 <7.4 × 10 –9 <7.4 × 10 –9 <2.5 × 10 –9 <2.5 × 10 –9 33 <7.4 × 10 –9 <7.4 × 10 –9 <2.5 × 10 –9 <2.5 × 10 –9 AZM <7.4 × 10 –9 <7.4 × 10 –9 <2.5 × 10 –9 <2.5 × 10 –9 AZI <7.4 × 10 –9 <7.4 × 10 –9 <2.5 × 10 –9 <2.5 × 10 –9 Rifampicin 1.06 × 10 –8 1.23 × 10 –8 3.6 × 10 –8 1.2 × 10 –8 Open in a new tab Chemoproteomics Target Engagement Evidence suggests that CA inhibition is the likely mechanism of action for the AZM-based anti-gonococcol activity, yet there is still a lack of target engagement data to support this or implicate other potential intracellular targets. Therefore, we utilized two probes that were designed and synthesized for target engagement studies in E. faecium () and applied them in the same chemoproteomics pull-down assays as an unbiased approach for identifying potential N. gonorrhoeae protein targets. The AZM-based pull-down probe Biotin-AZM consists of the AZM scaffold tethered to biotin via a PEG 8 linker. As a negative control pull-down probe, Biotin-Neg was designed in the same manner, except with the sulfonamide replaced by a methyl sulfone group. SAR has established that the sulfone analog does not inhibit the bacterial CAs nor exhibit any antimicrobial activity, likely through the loss of coordination to the Zn 2+ in the CA active site, making it a suitable inactive control for ruling out false positives. We confirmed that Biotin-AZM maintained binding to the N. gonorrhoeae CAs with K i values of 51.3 nM against the β-CA and 260 nM against the α-CA. The Biotin - Neg probe did not bind at concentrations up to 20 μM. The native expression level of each bacterial CA was quantified to understand relative expression using global label-free quantification (LFQ) proteomics. For this, we grew N. gonorrhoeae in overnight cultures, pelleted, washed, and lysed the cells in biological triplicate. Lysates were normalized to total protein concentration, then prepared for bottom-up proteomics with trypsin digestion followed by LC-MS/MS analysis. The LFQ intensities were normalized to the protein concentration in each sample. The data showed that the α-CA isoform has significantly greater expression, with 7.3 × 10 7 normalized LFQ intensity, compared to the β-CA, which has a 2.7 × 10 7 normalized LFQ intensity (). The biotin-functionalized probes were then employed in a pull-down assay with magnetic streptavidin beads to enrich target proteins from N. gonorrhoeae lysates in biological triplicate. Prior to the pull-down, N. gonorrhoeae FA1090 was grown, pelleted, and mechanically lysed via bead-beating. Lysates were cleared using centrifugation and normalized to 1.0–1.5 mg/mL total protein content. The magnetic streptavidin beads were incubated with 20 μM Biotin-AZM or Biotin-Neg for one hour, after which they were also incubated with biotin for additional blocking of streptavidin binding sites. Baseline control samples were also prepared, in which streptavidin beads were treated with DMSO rather than probe, to evaluate non-specific protein binding to the beads. The lysates were then added to the probe-treated and DMSO-treated streptavidin beads and incubated for three hours at 4 °C. Lysate supernatants were removed, and the beads were washed three times with PBS (pH 7.4) and twice with 0.1 M glycine (pH 2.4) to elute weak binders. Following these wash steps, the beads with their remaining bound target proteins were subjected to standard bottom-up proteomics sample preparation steps with on-bead trypsin digestion, followed by LC-MS/MS analysis. Peptides detected were matched to their corresponding proteins to identify targets and were quantified based on normalized abundances. Both Biotin-AZM and Biotin-Neg data sets were compared to the DMSO control data set to account for nonspecific binders to streptavidin Overall, 1,391 total proteins were identified across all treatment groups (full data set provided in ), and the data were visualized in volcano plots based on the Log 2 (fold-change) in protein abundance between probe-treated and DMSO-treated lysates and on the -Log 10 (p-value) for statistical significance in the pairwise analysis ( Figure ). Proteins enriched by a Log 2 (fold-change) ≥ 2.0 and -Log 10 (p-value) ≥ 1.3 were considered statistically significantly enriched by 4-fold or more in the Biotin-AZM pull-down compared to the DMSO control pull-down. In these samples, the only protein candidate that met these criteria was N. gonorrhoeae α-CA, showing the greatest enrichment in the Biotin-AZM pull-down by a 3.43 Log 2 (fold-change) with a 2.71 -Log 10 (p-value) ( Figure A). Interestingly, the N. gonorrhoeae β-CA was not enriched in the Biotin-AZM samples, indicating it is not a primary target for the scaffold in N. gonorrhoeae . In the Biotin-Neg samples, neither was the N. gonorrhoeae α-CA enriched providing further confidence in the target engagement for the scaffold ( Figure B). 4. Open in a new tab Streptavidin pull-down assay volcano plots demonstrating enrichment of bound proteins to probes from N. gonorrhoeae lysates. N. gonorrhoeae α-CA labeled with a green circle with a black border, N. gonorrhoeae β-CA labeled with a red circle with a black border. and indicated by the green circle. ( A ) Protein enrichment in Biotin-AZM pull-down samples compared to DMSO control pull-down samples. ( B ) Protein enrichment in Biotin-Neg pull-down samples compared to DMSO control pull-down samples. Criteria for enriched proteins (-Log 10 ( p -value) ≥ 1.3; Log 2 (fold-change) ≥ 2) is depicted by dashed lines. Other proteins found to be enriched by Biotin-Neg labeled as magenta circles. ( C ) Streptavidin pull-down competition assay volcano plots from N. gonorrhoeae lysates after pre-incubation with AZM at 100 μM. ( D ) Streptavidin pull-down competition assay volcano plots from N. gonorrhoeae lysates after pre-incubation with 18 at 100 μM. Criteria for competed-off proteins (-Log 10 ( p -value) ≥ 1.3; Log 2 (fold-change) ≤ −2) are depicted by dashed lines. To further investigate N. gonorrhoeae α-CA engagement, competition pull-down experiments were carried out. For these experiments, N. gonorrhoeae lysates were pre-incubated with 100 μM of free inhibitor AZM or 18 prior to the pull-down to compete with Biotin-AZM for target binding. If both inhibitors engage the same targets as Biotin-AZM , a loss of target proteins would be expected when comparing competition pull-down sample groups to the standard Biotin-AZM pull-down condition utilized above. Therefore, for the competition pull-down studies, the baseline control sample group for comparison consisted of lysates pre-incubated with DMSO rather than the inhibitor and subjected to pull-down with Biotin-AZM. Criteria determining the proteins that were successfully competed off from the pull-down probe by inhibitors were defined as proteins with a Log 2 (fold-change) ≤ −2 and -Log 10 (p-value) ≥ 1.3. The results from the pull-down competition experiments indicated that AZM and 18 both significantly compete with the N. gonorrhoeae α-CA for the Biotin-AZM probe. In the case of AZM, the only protein to be negatively enriched, with Log 2 (fold-change) of −3.39 was the N. gonorrhoeae α-CA when compared to the standard Biotin-AZM pull-down using N. gonorrhoeae lysates pre-treated only with DMSO ( Figure C). Similar results were obtained for the samples pre-incubated with 18, yielding a negative enrichment Log 2 (fold-change) of −4.68. An additional protein, biotin carboxyl carrier protein (Uniprot accession # Q5FAH5 ), was negatively enriched and met the same criteria in the 18 pre-incubation samples. This protein was not part of the enriched set for Biotin-AZM probe and may not be relevant to the target for antimicrobial activity, although further assessment is required. Taken collectively, with the observation that the anti-gonococcal activity of the analogs is dependent on the absence of elevated CO 2 , the data here suggest that α-CA is the primary intracellular target of the AZM scaffold and that its inhibition elicits an antimicrobial effect in N. gonorrhoeae . In Vivo Mouse Pharmacokinetics Studies Next, analogs 18 and 33 were evaluated for in vivo pharmacokinetic analysis in mice. BALB/c mice (n = 6 for each timepoint, 3 female and 3 male) were treated with either 18 or 33 at 20 mg/kg per os (p.o), and samples were taken at 0.167, 0.5, 1, 2, 4, 6, 8, and 24 hours to determine plasma concentrations over 24 hours ( Figure ). The compounds were at peak plasma fraction unbound concentrations (C p,u,max ) of 1.78 ± 0.64 μM ( 18 ) and 2.69 ± 0.56 μM ( 33 ) by the first timepoint ( Table ). Analog 33 also exhibited a slightly greater unbound area under the curve at 24 hours (AUC p,u,0 → 24 ) at 2.43 ± 0.18 h*μM compared to 18 with 2.15 ± 0.66 h*μM. The compounds were nearly equal in rates of clearance at approximately 63 and 68 mL/min/kg for 18 and 33 , respectively, but largely differed in their volumes of distribution (V z ) at 49.39 and 5.23 L/kg, respectively. Based on the PK data using the 20 mg/kg p.o. dose and the MIC against WHO-X, the pharmacodynamic indices for 18 were: C p,u,max /MIC = 8.9, AUC p,u,0 → 24 /MIC = 10.75, and time of unbound fraction concentration > MIC 10.75 and time of unbound fraction concentration > MIC (Tp,u > MIC) = 2.06 ( Table ). Comparatively, those values for 33 were Cp,u,max/MIC = 3.3, AUCp,u,024/MIC = 2.96, and Tp,u > MIC = 0.99 ( Table ). Two caveats should be noted in terms of the PK/PD data and how it may relate to in vivo efficacy. The first is that the concentrations represent plasma exposure, and this may not be fully relevant to urine or urethra exposure levels at the N. gonorrhoeae site of infection. Further PK assessment of these tissues would be warranted for a full picture of drug exposure to the relevant sites. The second is that these values will differ when dose-optimization studies are carried out, as an optimized efficacious dosing regimen, coupled with the relevant PK for that regimen, would provide more complete data with respect to the PK/PD indices and which PD metric drives the antibiotic effect. Nonetheless, for proof-of-concept evaluation and to select a candidate to move forward in efficacy assays, the current data still provides a framework for making that selection. Thus, based on these PK/PD values, we chose to move forward with analog 18 for evaluation in a urogenital model for N. gonorrhoeae infection. 5. Open in a new tab Total molecule concentration in plasma (C p ) and unbound molecule concentration in plasma (C p,u ) for 18 ( A ) and 33 ( B ) in mice (n = 6 for each timepoint; 3 female and 3 male) after a 20 mg/kg p.o. dose plotted over time. C p,u calculated using the mouse fraction unbound for each molecule presented in Table . MICs for each molecule versus N. gonorrhoeae WHO-X shown by a dotted black line. Error bars represent standard deviation (S.D.). Light gray box indicates the estimated range of MICs from the panel in Table . 9. In Vivo Pharmacokinetic Properties of 18 and 33 in Mice. T max C p,max C p,u,max AUC p,0 → 24 AUC p,u,0 → 24 V z CL p C p,u,max /MIC AUC p,u,0 → 24 /MIC T p,u > MIC Cpd (h) (μM) (μM) (h*μM) (h*μM) (L/kg) (mL/min/kg) (h) (h) 18 0.167 13.71 ± 4.88 1.78 ± 0.64 16.54 ± 5.08 2.15 ± 0.66 49.39 63.43 8.90 10.75 2.06 33 0.167 17.93 ± 3.88 2.69 ± 0.58 16.20 ± 1.26 2.43 ± 0.18 5.23 67.77 3.28 2.96 0.99 Open in a new tab a Mice were dosed at 20 mg/kg p.o., n = 6 for each timepoint with ± standard deviation. b Tmax is the time required to reach maximum concentration. c p,max = maximum plasma concentration. d C p,u,max = maximum unbound plasma concentration. e AUC p,0–24 = area under the curve until 24 h. f AUC p,u,0–24 = unbound fraction area under the curve until 24 h. g V z = volume of distribution at the terminal phase of elimination. h CL p = plasma clearance rate. i TMIC = length of time the concentration of the compound in the plasma unbound fraction remained above the MIC. In Vivo Mouse Model for Gonococcal Infection Analog 18 was prioritized for in vivo efficacy studies in a mouse model of N. gonorrheae infection. For the infected mouse model experiment, ovariectomized BALB/c mice were infected intravaginally with N. gonorrheae WHO-X and subsequently treated two days post-infection with 18 at 20 mg/kg (p.o.) bis in die (b.i.d., twice daily) for three consecutive days. The current drug of choice for N. gonorrheae infection, ceftriaxone, served as the positive control and was provided as a single dose at 15 mg/kg via the intraperitoneal (ip) route. Vehicle (10% DMA:20% propylene glycol:40% polyethylene glycol 400:30% PBS) was dosed orally as the negative control. In a previous study, AZM dosed once daily at 50 mg/kg, and was found to reduce gonococcal bioburden after three days. The dose regimen was based on its antigonococcal activity of 18 and the PK study showing that the unbound plasma concentration remains above the MIC for 2 hours. We opted for a twice-daily dosing regimen to maintain more exposure over the course of the study. After one day of treatment, mice treated with 18 had an approximately 1.5-log 10 (96.84%) CFU/mL reduction in gonococcal burden compared to vehicle-treated mice ( Figure ). Gonococcal burden continued to decrease in these mice, and by day 3, there was a 1.72-log 10 (98.1%) CFU/mL reduction of N. gonorrhoeae in mice treated with 18 ( Figure , Table ). This was comparable to the ceftriaxone-treated mice, which exhibited a slightly greater decrease in gonococcal burden at −2.09 Log 10 (99.2%) CFU/mL and cleared the infection to below the limit of detection in four mice, and 2 mice remained colonized with N. gonorrhoeae (∼10 4 –10 5 CFU/mL). This wide degree of variability in bacterial count within the ceftriaxone group may be attributed to that N. gonorrhoeae WHO-X is resistant to ceftriaxone, so the dose administered was able to clear the infection in some mice while others remained colonized. This initial experiment serves as proof of concept that improved AZM-scaffold bacterial CA inhibitors are capable of significantly reducing N. gonorrhoeae bioburden in a mouse model for infection after oral dosing. 6. Open in a new tab Log 10 CFU/mL of N. gonorrhoeae WHO-X (mean ± standard deviation) after treatment with 18 (20 mg/kg, p.o., b.i.d.) administered orally for three consecutive days. Ceftriaxone (CEF) (15 mg/kg, i.p., single dose) was included as a positive control, while the vehicle (10% DMA:20% propylene glycol:40% polyethylene glycol 400:30% PBS) served as a negative control. The dashed line indicates a lower limit of detection for CFU/mL counts. CFU/mL counts in the vaginal samples were analyzed (as compared to the vehicle control for each day and as compared to the vehicle control count on Day 0) via two-way ANOVA with post-hoc Dunnett’s test for multiple comparisons (n = 6 per group). **­( P < 0.01), *** ( P < 0.001). 10. N. gonorrhoeae CFU Counts for Mice on Day 3 a . Vehicle 18 Ceftriaxone Log 10 CFU/mL N. gonorrhoeae WHO-x Day 0 5.60 ± 0.23 4.92 ± 0.51 5.36 ± 0.38 1 5.53 ± 0.39 3.92 ± 0.62 3.86 ± 0.75 2 4.98 ± 0.66 3.16 ± 0.73 3.14 ± 0.95 3 5.10 ± 0.57 3.38 ± 0.76 3.01 ± 1.61 Δ Log 10 CFU/mL Day 3 compared to vehicle n.a .b –1.72 –2.09 Δ Log 10 CFU/mL Day 3 compared to vehicle at Day 0 –0.4 –2.22 –2.59 Open in a new tab Discussion The results presented in this study describe a SAR campaign based on the AZM scaffold, resulting in the development of 18 , an analog that exhibited favorable in vitro and in vivo PK properties and ultimately reduced gonococcal bioburden in an infected mouse model by approximately 98%. These findings build upon our previous work, which established that functionalizing the scaffold of AZM off its amide moiety is an effective strategy for improving both enzyme inhibition and antimicrobial activity. While that research resulted in two lead compounds, we herein aimed to test a larger cohort of analogs to improve our understanding of the SAR and further investigate top-performing compounds as viable drug leads. Furthermore, an initial study investigating in vivo efficacy revealed that AZM reduces N. gonorrheae bioburden in mice. On the basis of these data, we hypothesized that compounds with both improved in vitro potency against the bacteria, combined with evaluation of in vivo exposure, would result in improved efficacy in vivo as well. Dosing our lead analogs in mice for in vivo PK evaluation advanced our understanding of how AZM-based analogs will perform as drugs, as previous analogs were only investigated in an in vitro context. Additionally, the finding that analog 18 significantly reduced bioburden in mice at levels comparable to ceftriaxone, while acting through a novel mechanism of action in N. gonorrheae , is a significant finding in the pursuit of developing new antibiotics to address the ever-growing problem of antibiotic resistance. First, the comparison of 68 new analogs in our SAR study informed us about the moieties that are favorable for potent anti-gonococcal activity, providing insight into how to improve molecule design as we continue seeking to develop new therapies to treat gonorrhea. Evaluating drugs that were not potent also provided significant insight into our work developing CA inhibitors for N. gonorrhoeae , as identifying moieties that were detrimental to antimicrobial activity will also help steer future drug design. In general, cyclohexyl and phenyl derivatives with one to two methylenes removed from the amide increased potency compared to AZM. The addition of small substituents to these rings, like a methyl or fluorine, is also tolerated. While compounds with bridged bicyclic R-groups ( 27 and 28 ) retained potent activity, adding more steric bulk in the form of adamantyl groups and spirocycles, as well as replacing aliphatic rings with heterocycles, resulted in diminished activity. Two lead compounds, which had potent MICs (≤1 μM), were prioritized for further study to gain insight into their PK properties. While ceftriaxone, the current drug of choice for treating gonorrhea, is administered via intramuscular injection, we aimed to develop drugs that would be orally bioavailable. There are numerous advantages to orally dosed drugs, including patient compliance and ease of use. However, there are also several challenges to overcome when developing orally bioavailable drugs: they must be well-absorbed, water-soluble, and metabolically stable. We were therefore pleased to find that compounds 18 and 33 were stable in mouse liver microsomes, moderately soluble in aqueous and physiological solutions, and had logD 7.4 values within the agreement of a range typically observed in bioavailable drugs. Measuring absorption in a Caco-2 permeability assay revealed that the cyclohexyl-containing analogs have favorable P app values with ERs <2. Evaluating the plasma concentration of 18 in mice further illuminated that CA inhibitors have the potential to be a new class of compounds used to combat gonococcal infection. Although we developed lead compounds with potent enzyme inhibition and improved antimicrobial effects, one observation we noted was a lack of correlation when comparing antimicrobial data to enzyme inhibition data. The observation that potent K i s do not necessarily track with potent MICs has been remarked on by our group previously, regarding the SAR we have investigated for both AZM and EZM analogs. , With this lack of correlation, there could be several reasons. One is that we did not know the intracellular target of the scaffold, whether or not the molecules were inhibiting one or both bacterial CAs. To address this, a chemoproteomics study was carried out and identified that the primary target is likely the α-CA. A second, and perhaps more influential, reason for a disconnect between in vitro K i and MIC values may be differences in molecule accumulation in the Gram-negative pathogen. Small molecules that are able to permeate the cell membranes may be susceptible to efflux, leading to only a small concentration of the drug remaining inside the cell. , Studies from Hergenrother et al. , have identified physicochemical properties in small molecules that are attributed to permeability and accumulation in E. coli and P. aeruginosa , though to date no work has identified properties that affect molecule accumulation in N. gonorrhoeae . Our team has previously reported that EZM accumulates at significantly higher concentrations in N. gonorrhoeae than AZM, likely contributing to its 16-fold advantage in potency. It is therefore plausible that molecules in our SAR studies showing poor activity despite potent NgCA K i data may suffer from low accumulation, preventing the molecules from reaching their intracellular targets. Regardless, MIC potency drove the SAR design and prioritization for further in vitro antimicrobial assays, ADMET, and in vivo PK. Ultimately, analog 18 reduced N. gonorrheae bioburden in mice by 1.72-log 10 CFU within three days, while ceftriaxone, utilized as the positive control, reduced bioburden by approximately 2-log 10 CFU. However, examining the reduction in the bioburden of each individual mouse provides a clearer picture of efficacy between the two drugs. Treatment with 18 resulted in a reduction in the bioburden for all six mice in the group, with CFU counts all below the 10 5 CFU/mL range. Another unique advantage bacterial CA inhibition may have over currently prescribed treatment options is that the mechanism of action appears to be narrow-spectrum, only covering Enterococcus and N. gonorrhoeae species in our studies. It has been demonstrated that ceftriaxone significantly disrupts gut microbiome homeostasis in mice and humans, and treatment is an indicator of increased risk for Clostridioides difficile infection in patients due to disruption of the gut microbiome. ,, Similarly, patients treated with ceftriaxone have an increased risk for vaginal candidiasis due to perturbations in the vaginal microbiome. , Our group previously demonstrated that the bacterial CA inhibitor in the same scaffold class, CAI0019, spares the microbiome in mice when dosed orally at 20 mg/kg for 14 consecutive days, and this feature is likely due to the bacterial CA mechanism of action. It is unclear if this would extend to sparing the vaginal microbiome from dysbiosis, but the mechanism of action hypothesis suggests it would. Since 18 shares the same scaffold and mechanism of action as CAI0019, we hypothesize the molecule will also share the potential added benefit of little-to-no impact on the gut microflora. However, further studies on 18 ’s impact on gut microbiome balance after oral dosing are needed to test this hypothesis. Such a benefit could be impactful for improved health outcomes in patients with reduced complications arising from ceftriaxone treatment. Taken together, the proof-of-concept efficacy and potential to spare the microbiome are an advantage of 18 and further validate bacterial CA inhibition as a viable antibiotic strategy and a step forward in our drug discovery pipeline to combat N. gonorrhoeae . Conclusions Through this work, we have developed a thorough SAR for AZM-based compounds that exhibit a range of antimicrobial activity against N. gonorrhoeae and inhibition toward α- and β-NgCA. While analogs containing bulky hydrophobic six-membered rings continued to be among the most potent molecules, aligning with results from our previous study, the addition of heterocyclic moieties such as piperidines and tetrahydropyrans resulted in diminished antimicrobial effects. The SAR study culminated in the selection of 18 and 33 as lead analogs for further testing based on their potent MICs and enzyme inhibition. Additionally, we carried out the first chemoproteomic analysis of target engagement for this scaffold in N. gonorrhoeae which indicated the α-CA as the primary intracellular target. In vitro ADME evaluation of these analogs led us to prioritize 18 and 33 for in vivo PK, wherein analog 18 was revealed to have better antibiotic PK/PD indices than 33 . These properties translated to efficacy in the infected mouse model, as 18 reduced gonococcal burden by 98% compared to the vehicle within three days of treatment. This data, paired with the low cytotoxicity against human cell lines and low spontaneous mutation frequency, demonstrates the potential of lead compound 18 to be a viable therapeutic for N. gonorrhoeae . The prospect of an orally bioavailable, microbiome-sparing drug with a novel mechanism of action is a promising step toward combating increasing rates of gonorrhea infections. Future directions will focus on an optimized dosing regimen and longer in vivo efficacy studies to better understand how this potent CA inhibitor can be further developed into an anti-gonococcal agent. Experimental Chemistry: General Methods 1 H and 13 C NMR spectra were recorded on either a Bruker DRX500 (operating at 500 and 126 MHz) in DMSO-d 6 with the internal standard of TMS at 0.05% v/v. Chemical shifts (δ) are reported as parts per million (ppm), and the coupling constants are reported as s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet, m = multiplet. The purity of all final compounds was >95%, as assessed by HPLC using an Agilent 1200 series chromatograph. The chromatographic method utilized a ThermoScientific Hypersil GOLD C-18 column. UV detection wavelength = 220/254 nm, flow-rate = 1.0 mL/min, and mobile phases of (1) acetonitrile/water with 0.1% formic acid. An Advion CMS-L compact mass spectrometer with an APCI source and an atmospheric solids analysis probe (ASAP) was used for mass spectrometry analysis. AZM (A2B Chem, Catalog no. AB70385) was purchased from a commercial vendor, then fully characterized to confirm its identity and purity prior to biological evaluation. Most analogs were previously reported for activity against Enterococcus , with synthetic routes for compounds 71 and 72 previously reported with experimental for both Enterocococcus and N. gonorrhoeae activity against the CDC 181 strain. The same samples of previously reported analogs were utilized for evaluation against N. gonorrhoeae in this study. Therefore, experimental and spectroscopic data can be found in publications by Holly et al. Experimental and characterization data for analog 58 and resynthesized batches of 18 and 33 that were used in in vivo studies, are reported here. Synthesis 5 - Amino-1,3,4-thiadiazole-2-sulfonamide ( 4a ) To a round-bottom flask was added acetazolamide (5.0 g, 22.0 mmol, 1 equiv) in 12 M HCl (56 mL, 670 mmol, 30 equiv). The solution was stirred at 95 °C for 16 h, then allowed to cool, placed on ice, and slowly quenched with 5 M NaOH until the solution reached neutral pH. The mixture was concentrated using rotary evaporation, and the resulting white solid was dissolved in acetone and filtered. The filtrate was concentrated to yield the final product as a white solid (3.04 g, 16.89 mmol, 75%). 1 H NMR (500 MHz, DMSO-d 6 ): δ 8.02 (s, 2H), 7.77 (s, 2H). APCI-MS: m/z 180.9 [M + H] + . 2-(3-Methylcyclohexyl)-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)­acetamide ( 18 ) Step 1. To a vial 2-(3-methylcyclohexyl)­acetic acid (0.225 g, 1.44 mmol, 1.3 equiv) was added in DCM (2.0 mL). This was cooled to 0 °C, followed by the addition of DMF (2 drops) and oxalyl chloride (0.183 g, 1.44 mmol, 1.3 equiv). The reaction was then allowed to warm to room temperature and stirred for 2.5 h. After 2.5 h, the reaction was concentrated in vacuo, and the crude acyl chloride was carried into step 2 uncharacterized. Step 2. In an argon-flushed vial 4a (0.20 g, 1.11 mmol, 1 equiv) dissolved in 7.0 mL of acetonitrile, followed by potassium carbonate (0.215 g, 1.55 mmol, 1.4 equiv) at 0 °C. Crude acid chloride (from Step 1) was then diluted with acetonitrile (3.0 mL) and added dropwise to the reaction mixture at 0 °C. The reaction mixture was stirred for 16 h at rt, then concentrated in vacuo . Water (20 mL) was added to the obtained residue, and the solution was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo. The obtained residue was purified by reverse-phase chromatography (gradient elution with 0–40% acetonitrile in water) to afford the product as a white solid (0.056 g, 0.18 mmol, 16%). 1 H NMR (500 MHz, DMSO- d 6 ) δ: 13.01 (s, 1H), 8.33 (s, 2H), 2.45–2.35 (m, 2H), 2.22–2.14 (m, 0.17H), 1.85–1.75 (m, 1H), 1.70–1.58 (m, 4H), 1.55–1.42 (m, 0.76H), 1.38–1.30 (m, 1H), 1.27–1.18 (m, 1H), 1.10–1.05 (m, 0.18H), 0.90–0.73 (m, 5H), 0.66–0.57 (m, 1H). 13 C NMR (126 MHz, DMSO- d 6 ) δ: 171.9, 171.6, 164.3, 161.1, 42.7, 41.2, 38.3, 34.7, 34.4, 33.1, 32.0, 30.7, 29.7, 26.7, 25.6, 22.8, 20.7, 20.3. APCI-MS: m/z 318.5 [M+H] + . HPLC retention time (mixture of cis and trans diastereomers): 7.458 min & 7.508 min. HPLC Purity: >99%. Elemental analysis- Theoretical: C, 41.49; H, 5.70; N, 17.60; S, 20.14. Found: C, 41.39; H, 5.68; N, 17.38; S, 20.15. 3-Methyl-N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)­cyclohexane-1-carboxamide ( 33 ) Step 1. To a vial was added 3-methylcyclohexane-1-carboxylic acid (0.100 mg, 0.703 mmol, 1.3 equiv) and thionyl chloride (0.50 mL). This was heated to 60 °C and stirred for 2.5 h. After 2.5 h, the reaction was concentrated in vacuo, and the crude acyl chloride was carried into step 2 uncharacterized. Step 2. n an argon-flushed vial 4a (0.10 g, 0.555 mmol, 1.0 equiv) dissolved in 7.0 mL of acetonitrile, followed by triethylamine (0.11 mL, 0.777 mmol, 1.4 equiv) at 0 °C. Acid chloride (from Step 1) was then diluted with acetonitrile (3.0 mL) and added dropwise to the reaction mixture at 0 °C. The reaction mixture was stirred for 16 h at rt, then concentrated in vacuo . Water (20 mL) was added to the obtained residue, and the solution was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated in vacuo . The obtained residue was purified by reverse-phase chromatography (gradient elution with 0–40% acetonitrile in water) to afford the final product, isolated as a white solid (0.073 g, 0.24 mmol, 43%). 1 H NMR (500 MHz, DMSO- d 6 , mixture of diastereomers) δ: 12.96 (s, 1H), 8.33 (s, 2H), 2.90–2.84 (m, 0.3H), 2.64–2.54 (m, 0.7H), 1.91–1.72 (m, 3H), 1.72–1.60 (m, 1H), 1.58–1.47 (m, 1H), 1.45–1.36 (m, 1H), 1.34–1.21 (m, 1H), 1.18–1.14 (m, 0.4H), 1.11–1.02 (m, 7H), 0.94–0.80 (m, 4H). 13 C NMR (126 MHz, DMSO- d 6 , mixture of diastereomers) δ: 175.2, 175.0, 164.4, 164.3, 161.3, 161.3, 43.5, 38.5, 37.0, 34.9, 33.9, 32.4, 31.4, 28.3, 27.8, 27.2, 25.0, 22.6, 20.6, 20.1. APCI-MS: m/z 304.9 [M+H] + . HPLC retention time: 7.118 min. HPLC Purity: 97.3%. 5-((2-Cyclohexylethyl)­amino)-1,3,4-thiadiazole-2-sulfonamide ( 58 ) To a vial was added 4a (0.224 g, 1.24 mmol, 1 equiv) and 2-cyclohexylacetaldehyde (0.226 g, 1.79 mmol, 1.44 equiv) in anhydrous DCM (4 mL) and anhydrous methanol (4 mL) under argon. Acetic acid (0.004 mL, 0.062 mmol, 0.05 equiv) was added at rt, and the reaction was allowed to stir for 1 h. Sodium cyanotrihydroborate (0.124 g, 1.97 mmol, 1.58 equiv) was dissolved in methanol (3 mL) and added to the reaction mixture dropwise at 0 °C. The mixture was allowed to warm to rt and stirred for 16 h. The reaction was quenched with water and extracted with DCM (3 × 30 mL). The combined organic layers were dried over sodium sulfate, then concentrated in vacuo . The obtained residue was purified by reverse-phase chromatography (gradient elution with 0–40% acetonitrile in water) to afford the final product as a white powder (0.130 g, 0.447 mmol, 36%). 1 H NMR (500 MHz, DMSO- d 6 ) δ: 8.29 (t, J = 5.6 Hz, 1H), 7.91 (s, 2H), 3.0–2.96 (m, 2H), 1.67–1.53 (m, 5H), 1.36–1.21 (m, 3H), 1.21–1.02 (m, 3H), 0.87–0.75 (m, 2H). 13 C NMR (126 MHz, DMSO- d 6 ) δ: 171.85, 155.33, 40.61, 36.40, 34.05, 32.47, 26.07, 25.71. APCI-MS: m/z 291.2 [M+H] + . HPLC retention time: 7.985 min. HPLC Purity: 96.4%. Biological Evaluation Bacterial Strains, Media, and Chemicals N. gonorrhoeae strains used in the study were obtained from the American Type Culture Collection (ATCC), CDC, and WHO. The Vero and ME-180 cell lines were obtained from the ATCC. Media and reagents used in this work were purchased commercially: azithromycin and ceftriaxone (TCI America); Minimum Essential Medium (MEM), McCoy’s 5A medium, and hematin (Sigma Aldrich); Triton X-100 (Acros Organics); nicotinamide adenine dinucleotide (NAD) (Chem-Impex International); MTS (3-(4,5-dimethylthia-zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Abcam); and brucella broth, Chocolate II agar plates, IsoVitaleX, and bovine hemoglobin (Becton, Dickinson and Company). Carbonic Anhydrase CO 2 Hydration Assay and K i Determination The assay was performed as previously described. , Recombinant N. gonorrheae CAs were produced as previously described; recombinant human CAs I and II were purchased from Millipore Sigma (hCAI Catalog # C4396-5MG; hCAII Catalog # C6624-500UG). K i values were calculated by inputting IC 50 values into the Cheng-Prusoff equation for K i from catalytic inhibition constants. Antibacterial Activity of Analogs Against N. gonorrhoeae The MICs of AZM and its analogs were determined using a broth microdilution assay, as previously described. ,, N. gonorrhoeae strains were grown overnight on Chocolate II agar plates. A bacterial solution equivalent to a 1.0 McFarland standard was prepared and diluted in Brucella broth supplemented with yeast extract, dextrose, proteose-peptone, NAD, pyridoxal, hematin, and IsoVitaleX to reach a bacterial count of about 1 × 10 6 CFU/mL. Test agents were serially diluted along the plates. Media containing bacteria (without test agents) were included in the assays as a growth control. Plates were then incubated aerobically and in the presence of 5% CO 2 for 24 h at 37 °C prior to recording the MIC, as observed visually. Affinity Pull-Down of Target Proteins from the N. gonorrhoeae FA1090 Lysate N. FA1090 Lysate Preparation N. gonorrhoeae FA1090 cultures were prepared in biological triplicates by inoculating 5 mL cultures of tryptic soy broth (TSB), each with a single colony grown on tryptic soy agar. Cultures were incubated at 37 °C for 20 h with shaking at 250 rpm. Subcultures were then prepared by inoculating 100 mL TSB with 2 mL (1:50 dilution) of overnight culture and incubating at 37 °C with shaking at 250 rpm until mid-logarithmic growth was reached (OD 600 = 1.0). Cultures were then centrifuged at 4,000 × g for 20 min to pellet. The supernatant was discarded, and the pellets were subjected to three washes with 1× PBS, pH 7.4, with centrifugation at 4,000 × g for 5 min after each wash to pellet. After the final wash, the supernatant was discarded, and the pellet was resuspended in cold lysis buffer (1× TBS, 1 mM DTT, 1× Halt Protease Inhibitor, 0.001% w/v Rapigest TM SF, Waters Corp.) at a 1:5 ratio of pellet mass (g) to buffer volume (mL). Lysozyme was also added to the lysis suspension at a final concentration of 0.2 mg/mL to aid in peptidoglycan breakdown. The lysis suspension was transferred to Precellys® VK05 tubes and homogenized using a Precellys®24 Touch (Bertin Corp.) tissue homogenizer (6,000 rpm; 6 cycles of 15 s on, 30 s off). The lysate was clarified by centrifugation at 5,000 × g for 10 min to pellet debris. The lysate supernatant was collected, and protein concentration was quantified using the Pierce TM BCA Assay Kit (ThermoFisher). Total protein concentration was adjusted to 1.0–1.5 mg/mL in lysis buffer prior to pulldown experiments. Biotin-AZM and Biotin-Neg Pull-Downs from the N. gonorrhoeae Lysate Pull-down experiments were performed in biological triplicate with N. gonorrhoeae FA1090 lysates in accordance with the following bead treatment groups: DMSO (negative control), Biotin-AZM , and Biotin-Neg . Biotin-AZM and Biotin-Neg were prepared as 10 mM solutions in DMSO. Samples containing 50 μL of Pierce Streptavidin Magnetic Beads (ThermoFisher) were aliquoted in triplicate for each treatment group and washed three times with 150 μL of TBSR wash buffer (20 mM Tris, 0.15 M NaCl, pH 7.4, 0.001% w/v Rapigest), sequestered with a magnetic stand, and the supernatant was discarded after each wash. Solutions of 0.2% DMSO in wash buffer, 20 μM Biotin-AZM in wash buffer, and 20 μM Biotin-Neg in wash buffer were prepared from the respective DMSO stock solutions. Aliquots of 100 μL of 0.2% DMSO, 20 μM Biotin-AZM , and 20 μM Biotin-Neg were each added to their respective beads and incubated at 4 °C for 2.5 h with rotation. Post-incubation, the probe supernatant was discarded from the beads, and 100 μL of 2 mM D-biotin blocking buffer solution in wash buffer was added to the beads and incubated at room temperature for 5 min off the magnetic rack. The beads were sequestered using the magnetic rack, and the supernatant was discarded. The biotin blocking step was repeated one additional time. The beads were then subjected to two additional wash steps with 150 μL of wash buffer. 100 μL of 1.0–1.5 mg/mL pre-incubated N. gonorrhoeae lysate was added to the beads and incubated at 4 °C for 3 h with rotation. The supernatant was discarded, and the beads were washed with 100 μL of wash buffer. The supernatant was discarded and the beads were washed a second and third time with wash buffer containing increasing concentrations of sodium chloride (second wash: 20 mM Tris, 0.5 M NaCl, pH 7.4, 0.001% w/v Rapigest TM ; third wash: 20 mM Tris, 1.0 M NaCl, pH 7.4, 0.001% w/v Rapigest TM ). The beads, elutes, and untreated input lysates (100 μg from each biological replicate) were stored at −80 °C until they were ready to be prepped for on-bead digestion at the proteomics core. Competition Pulldown with AZM and 18 Competition pulldown experiments were performed in biological triplicate as follows. The inhibitors AZM and 18 were prepared as 10 mM solutions in DMSO. The Pierce Streptavidin Magnetic Beads were prepped as described above. Prior to the pulldown, N. gonorrhoeae lysates were pre-treated with DMSO (negative control), AZM, or 18 to a final concentration of 1% DMSO or 100 μM inhibitor and incubated at 4 °C for 2 h with rotation to allow for target binding. During lysate pre-treatment, the pulldown workflow with Pierce Streptavidin Magnetic Beads was begun as described above, using 20 μM Biotin-AZM as the probe for all samples. After the probe incubation and biotin-blocking steps, 100 μL of each pre-treated lysate was added to the beads and incubated at 4 °C for 3 h with rotation. The supernatant was discarded, and the remaining wash and elution steps were performed as described above. The beads, eluates, and untreated input lysates (100 μg from each biological replicate) were stored at −80 °C until further proteomics sample preparation could be completed. Sample Preparation for Bottom-Up Proteomics Analysis The samples were processed with standard on-bead bottom-up proteomics protocol as previously described. , Briefly, the beads were incubated with ammonium bicarbonate (20 mM) and kept under shaking with thermomixer at 800 rpm for 15 min at room temperature. Samples on beads were reduced using dithiothreitol (DTT) for 1 h at room temperature with shaking at 800 rpm. The samples on beads were alkylated using iodoacetamide (IAM) for 1 h in the dark at room temperature with shaking at 800 rpm. The residual IAM was quenched using DTT. The samples were then diluted with 600 μL of ammonium bicarbonate (20 mM) and the pH was adjusted to 7.4 for trypsinization. Approximately 1 μg of Trypsin/LysC mix was added to the samples and allowed for overnight digestion at 37 °C with shaking in a thermomixer at 800 rpm. The samples were subjected to another 0.5 μg of fresh Trypsin/LysC mix in the morning and allowed to digest for another 4 h. The reaction was stopped by adding trifluoroacetic acid (TFA). The residual particles were separated using a magnetic separator. The digested peptides were adjusted to pH < 3 using TFA and subjected to a Pierce C18 peptide desalting column (Thermo Fisher Scientific, 89852) following the manufacturer’s recommendations. Proteomic Data Acquisition and Processing Dried peptides obtained were reconstituted in 3% ACN and 0.1% formic acid (FA) before being analyzed by HPLC-MS/MS equipped with an Ultimate 3000 HPLC (Thermo Fisher Inc., Waltham, MA) and an Aurora TM Ultimate analytical column (IonOpticks, Victoria, AUS), as previously described. Approximately 0.6 μg of peptides were loaded onto a trap column (150 mm long × 75 μm ID, 3 μm particle size, 100 Å C18 medium) and separated on a 25 cm nanoflow UHPLC IonOpticks C18 column. A 130-min elution gradient was employed by mixing mobile phase solvent A (0.1% FA in water) with solvent B (80% ACN, 0.1% FA in water). The gradient method was carried out with percentages and terminal gradient time points as follows: 2–12% B (1.6 min), 12–25% (80 min), 25–35% (100 min), 35–45% (105 min), 45–95% (120 min), 95% hold (125 min), 95–2% (125.1 min), 2–5% (130 min). For each sample, 1 μg of sample peptide was injected. Data were acquired using an Orbitrap Fusion Lumos MS (ThermoFisher). The mass spectrometer data acquisition settings for the Lumos were followed as previously described. , The performance of the mass spectrometer was validated by injecting HeLa digests before and after the injection. Raw proteomics data were processed using the Proteome Discoverer (version 3.1, Thermo Fisher) software using N. gonorrhoeae FA1090 proteome (UniP rot). An in-house list of common contaminants was added to the search, and these were excluded from the final analysis. The raw data search was done following the manufacturer’s recommendations and the detailed settings as previously described. The final data were processed using RStudio, Excel, and Persues LogD Calculations The method and quantification were performed as previously described. Briefly, 10 μL solutions of each compound were pipetted into a pre-determined volume (0.5, 0.66, or 0.8 mL) of pre-saturated PBS (pH 7.4) and mixed well for 5 min. Solutions were centrifuged for 5 min, then pre-saturated 1-octanol (0.5, 0.33, or 0.2 mL) was added to yield a 1.0 mL solution of 1:1, 2:1, or 4:1 PBS:buffer. Solutions were equilibrated on an end-to-end rotator for 2 h, then centrifuged for 10 min. 200 μL aliquots of each organic phase and aqueous phase were subjected to HPLC analysis, using standard curves for quantitation. LogD was calculated by the equation LogD = log [ Organic layer ] [ Aqueous layer ] Caco-2 Permeability Assay Assays and data analysis were performed by Eurofins Panlabs (MO, USA) according to the procedure described by Hidalgo et al. Mouse Liver Microsome Stability Assays and data analysis were performed by Eurofins Panlabs (MO, USA) according to the procedure describef by Obach et al. Plasma Protein Binding Assays and data analysis were performed by Eurofins Panlabs (MO, USA) according to the procedure described by Banker et al. Solubility Compounds were tested for aqueous solubility in PBS, along with solubility in simulated gastric fluid (without pepsin) and simulated intestinal fluid (Ricca Chemical). DMSO stock solutions of each compound (50 mM) were diluted into each solution to achieve a range of mM concentrations containing 0.05% DMSO. Solutions were added to a 96-well plate alongside blanks of each corresponding solution (200 μL per well, n = 4) and read on a BMG LABTECH NEPHELOstar Plus to measure forward-scattered light, measured in counts. Plates were read vertically with a laser intensity of 90%, and raw data was blank-subtracted to obtain the solubility curve. Two linear lines were fitted to the data, and the point at which they cross (the highest concentration prior to a significant increase in counts) was taken as the kinetic solubility. Plasma Stability Compounds were tested for their stability in mouse plasma based on a previously established protocol. From a 5 mM DMSO stock solution, compounds were diluted to 50 μM in PBS, then added to a solution of 50:50 reconstituted mouse plasma:PBS (pH = 7.4). Plasma solution (315 μL) and each test agent (35 μL) were aliquoted into Eppendorf tubes (final concentration = 5 μM), and then the tubes were incubated with shaking at 37 °C. At each timepoint (0, 30, 60, 90, 120, 180 min), 50 μL were taken from each sample and quenched with 300 μL cold acetonitrile. The solutions were briefly vortexed and then centrifuged at 3000 rpm for 15 minutes. The supernatant (100 μL) was transferred to a vial and diluted 50% with water spiked with 0.2 μM AZM to be used as an internal standard. The samples were submitted for LCMS analysis. Percent remaining was calculated by the equation % remaining = 100 ( P A R @ t P A R @ t 0 ) where PAR is the peak area ratio of analyte to internal standard. In Vivo Pharmacokinetics Assays and data analysis were performed by Eurofins Panlabs (Taiwan). All aspects of the work were performed in general accordance with the Guide for the Care and Use of Laboratory Animals: Eighth Edition in an AAALAC-accredited laboratory animal facility. The animal care and use protocol was reviewed and approved by the Institutional Animal Care and Use Committee at Pharmacology Discovery Services Taiwan, Ltd. under institutional approval number PK001-08062024. BALB/c mice (n = 6 per time point per molecule tested) were dosed with the test compound at 20 mg/kg by oral gavage (PO) in vehicle (10% dimethylacetamide/20% propylene glycol/40% polyethylene glycol 400/30% phosphate-buffered saline). Blood aliquots were collected at 8 timepoints (0.167, 0.5, 1, 2, 4, 6, 8, and 24 h) after PO administration from each mouse. The first seven timepoint blood aliquots were collected via the facial vein (0.05 mL), and the final timepoint was collected via cardiac puncture (0.3 mL). Samples were then centrifuged at 2,500 × g for 15 minutes at 4 °C within 1 hour of collection. The plasma samples were processed using acetonitrile precipitation and analyzed by LC-MS/MS. The exposure levels (ng/mL) of all molecules in plasma samples were determined by LC-MS/MS. Plots of plasma concentrations (mean ± SD) vs. time for the molecule were constructed. The fundamental PK parameters (t 1/2 , T max , C max , AUC last , AUC Inf , AUC Extr MRT, Vz, and CL) after PO administration were obtained from the analysis of the plasma data using WinNonlin (best-fit mode). Antibacterial Killing Kinetics A standard time-kill kinetics assay was performed against N. gonorrhoeae WHO-X as described previously. − N. gonorrhoeae was grown in GC broth to the logarithmic phase and diluted to an inoculum of approximately 10 6 CFU/mL. Compounds were dosed at 5× MIC in triplicate and incubated in ambient air at 37 °C for 24 h. DMSO was used as the negative control. At each time point (0, 2, 4, 6, 8, 12, 24 h), an aliquot from each sample was serially diluted and plated onto Chocolate II agar plates. Plates were incubated at 37 °C for 24 h to calculate viable CFU/mL. Frequency of Resistance A single-step resistance assay was implemented as previously described − to test N. gonorrhoeae WHO-X against the compounds. Plates were prepared by mixing each agent (at 2×, 3×, 5×, and 10× MIC) with GC agar supplemented with IsoVitaleX, then allowed to dry at room temperature. A bacterial suspension of approximately 10 9 CFU/mL was spread onto the plates, which were incubated at 37 °C for 48 h and then counted to calculate CFU. In Vitro Cytotoxicity Compounds were dosed against monkey fibroblast (Vero) cells and human endocervical (ME-180) cells at concentrations in a dose-response up to 500 μM, as previously described. − Cells were incubated with each compound in triplicate in a 96-well plate for 24 h at 37 °C. DMSO was dosed at equal concentrations to the drug-treated cells to serve as the negative control. MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) (Promega) was subsequently added to the wells, and plates were incubated for up to 4 h. Absorbance readings (at OD 490 ) were recorded using a kinetic microplate reader. The number of viable cells after treatment was expressed as a percentage of the viability of the DMSO-treated cells. Data were analyzed via two-way ANOVA with post hoc Dunnett’s test for multiple comparisons. In Vivo Efficacy in a Mouse Model of N. gonorrhoeae Infection Animal housing and experiments in this study were reviewed, approved, and performed under the guidelines of the Virginia Polytechnic Institute and State University Animal Care and Use Committee, approval number IAUCUC-23–227 and were carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Mice were housed in individually ventilated cages (5 per cage) with free access to food and water. All mice were acclimatized for seven days before the start of the experiment. The mouse model of N. gonorrheae genital tract infection was performed as described previously., ,− Estradiol Implantation On day −2, ovariectomized 8-week-old female BALB/c mice were implanted with a 5-mg, 21-day controlled-release estradiol pellet subcutaneously using precision trocars. Afterward, a drop of tissue adhesive was applied, and the skin was pressed to close the wound. Antibiotic Administration Antibiotics were administered to increase the susceptibility to N. gonorrhoeae as previously described. , Mice were injected intraperitoneally with 0.6 mg of vancomycin and 1.2 mg of streptomycin on Days −2 through +1. The drinking water was replaced on Day −2 with sterilized water containing 0.4 g/L trimethoprim. Trimethoprim water was renewed every other day throughout the duration of the experiment. Streptomycin sulfate (2.4 g/L) was added to the trimethoprim water starting on Day +2 until the end of the experiment. N. gonorrhoeae Infection and Treatment Two days after pellet implantation (Day 0), the vagina of each mouse was rinsed with 50 mM HEPES (pH = 7.4), and mice were inoculated intravaginally with 2.38 × 10 6 CFU/mouse of streptomycin-resistant N. gonorrhoeae WHO-X ( stm R ) (each mouse was infected with 15 μL from the bacterial inoculum). Two days post-infection (Day +2), mice were randomly allocated into groups (n = 6). Mice were treated with test agents at a dose of 20 mg/kg twice daily for three consecutive days via oral gavage. As controls, mice were administered either the vehicle (10% dimethylacetamide:20% propylene glycol:40% polyethylene glycol 400:30% PBS), orally twice daily for three consecutive days, or a single intraperitoneal dose of CEF (15 mg/kg in water). Sample Collection and Counting of N. gonorrhoeae Vaginal swabs were collected daily by gently inserting a moistened Dacron swab into the vagina of anesthetized mice and suspending the swab in 0.1 mL of GC broth containing 0.05% saponin. Samples were serially diluted and plated on GC agar supplemented with vancomycin, colistin, nystatin, and trimethoprim. A small portion of the sample was also cultured on heart infusion agar to monitor the presence of potentially inhibitory commensal bacteria. Gram staining was performed to further identify the grown commensals (if any). If any mice were found to be colonized with enteric Gram-negative rods that might contribute to the clearance of N. gonorrheae , these mice were excluded from the experiment and the data analysis. Mice were humanely euthanized via carbon dioxide asphyxiation at the end of the experiment. Statistical Analysis Data was analyzed using GraphPad Prism version 9.0 for Windows (GraphPad Software, La Jolla, CA).The data of N. gonorrhoeae CFU counts in the vaginal samples were analyzed via two-way ANOVA with post-hoc Dunnett’s test for multiple comparisons ( P <0.05). Supplementary Material jm6c00012_si_001.pdf (1.6MB, pdf) jm6c00012_si_002.xlsx (314KB, xlsx) jm6c00012_si_003.xlsx (6MB, xlsx) jm6c00012_si_004.csv (5.2KB, csv) Acknowledgments The authors would like to acknowledge the research support and space provided by the Purdue College of Pharmacy to make this work possible. We thank the CDC and the FDA Antibiotic Resistance Isolate Bank, Atlanta, GA, for supplying several of the clinical isolates used in this study. The TOC graphic was created in BioRender. Flaherty, D. (2026) https://BioRender.com/7gaez4z . Glossary Abbreviations Used ADME Absorption, Distribution, Metabolism, Excretion AUC area under the curve CA carbonic anhydrase CFU colony-forming unit; Cl int , intrinsic clearance C max maximum concentration CO 2 carbon dioxide CYP cytochrome P450 DMSO dimethyl sulfoxide hCA human carbonic anhydrase MIC minimum inhibitory concentration NgCA N. gonorrhoeae Carbonic Anhydrase PBS phosphate-buffered saline SAR structure-activity relationship Vz volume of distribution at the terminal phase of elimination Proteomics data have been deposited in the MassIVE repository under the ID MSV000100717. The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.6c00012 . MIC values for strains in 5% CO 2 ; correlation plots for α-NgCA Ki vs MIC; mammalian cell viability; structures of chemoproteomics probes; LFQ intensities for global proteomics on N. gonorrhoeae , compound characterization including 1 H- and 13 C-NMR spectra and mass spectra for all analogs, as well as HPLC chromatograms for selected analogs taken forward for in vitro ADME and in vivo studies all provided ( PDF ) Proteomic native expression data ( XLSX ) Chemoproteomic pull-down data ( XLSX ) Molecular formula strings with biochemical and MIC data for all analogs provided ( CSV ) Molecule design and synthesis was by P.R.T., M.S.Y., K.J.H., and D.P.F. Collection of antibacterial data was done by N.S.A., F.A., A.S.A., and M.S.Y. Recombinant protein expression and purification was done by A.K.M. The in vitro ADMET experiments by M.S.Y., O.C.S., F.L.D., and D.P.F. In vivo pharmacokinetic analysis was done by M.S.Y. and D.P.F. Carbonic anhydrase inhibition by A.N. and C.T.S. Proteomics sample preparation was done by F.A. and C.K.M. Proteomics data collection and analysis was done by V.P.T. and C.-J.L. In vivo efficacy by N.S.A., A.S.A., and M.N.S. The manuscript was written by P.R.T., M.S.Y., and D.P.F., with editing and contributions by N.S.A., F.A., A.N., C.T.S., and M.N.S. All authors have given approval to the final version of this manuscript. P.R.T. and M.S.Y. gave equal contribution The work was funded by the Purdue College of Pharmacy and the Chip and Jane Rutledge Donation (D.P.F.), and the Purdue Institute for Drug Discovery Program Grant (D.P.F. and M.N.S.). Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Numbers T32AI148103, 1R01AI153264 (D.P.F. and M.N.S.), and 1R01AI148523 (D.P.F. and M.N.S.). 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